Alternative nucleic acid molecules containing reduced uracil content and uses thereof

ABSTRACT

The present disclosure provides alternative nucleosides, nucleotides, and nucleic acids, and methods of using them. In some aspects, the disclosure provides mRNA wherein the uracil content has been modified and which may be particularly effective for use in therapeutic compositions, because they may benefit from both high expression levels and limited induction of the innate immune response. In some aspects, the disclosure provides methods for the production of pharmaceutical compositions including mRNA without reverse phase chromatography.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

BACKGROUND OF THE INVENTION

There are multiple problems with prior methodologies of effectingprotein expression. For example, heterologous DNA introduced into a cellcan be inherited by daughter cells (whether or not the heterologous DNAhas integrated into the chromosome) or by offspring. Introduced DNA canintegrate into host cell genomic DNA at some frequency, resulting inalterations and/or damage to the host cell genomic DNA. In addition,multiple steps must occur before a protein is made. Once inside thecell, DNA must be transported into the nucleus where it is transcribedinto RNA. The RNA transcribed from DNA must then enter the cytoplasmwhere it is translated into protein. This need for multiple processingsteps creates lag times before the generation of a protein of interest.Further, it is difficult to obtain DNA expression in cells; frequentlyDNA enters cells but is not exprfressed or not expressed at reasonablerates or concentrations. This can be a particular problem when DNA isintroduced into cells such as primary cells or modified cell lines.

Naturally occurring RNAs are synthesized from four basicribonucleotides: ATP, CTP, UTP and GTP, but may containpost-transcriptionally modified nucleotides. Further, approximately onehundred different nucleoside alterations have been identified in RNA(Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA ModificationDatabase: 1999 update. Nucl Acids Res 27: 196-197).

There is a need in the art for biological modalities to address themodulation of intracellular translation of nucleic acids. The presentinvention solves this problem by providing new mRNA moleculesincorporating chemical alterations which impart properties which areadvantageous to therapeutic development.

SUMMARY OF THE INVENTION

The present disclosure provides, inter alia, alternative nucleosides,alternative nucleotides, and alternative nucleic acids including analternative nucleobase, sugar, or backbone.

In a first aspect, the invention features an mRNA encoding a polypeptideof interest and including an open reading frame, wherein (a) the uracilcontent of the mRNA is less than 20% of the total nucleotide content inthe open reading frame; and (b) at least 90% (e.g., at least 95%, atleast 99%, or 100%) of the uracils in the open reading frame are5-methoxy-uracil. In preferred embodiments, the open reading frameconsists of nucleotides including 5-methoxy-uracil and/or uracil,cytosine, adenine, and guanine.

In some embodiments, the uracil content of the mRNA is different thanthe uracil content of a corresponding wild-type sequence. In otherembodiments, the percentage of uracil of the total nucleotide content inthe open reading frame is different relative to the correspondingwild-type sequence. In certain embodiments, the percentage of uracils ofthe total nucleotide content in one or more subsequences (e.g., asubsequence 5 to 40 nucleotides in length) of the open reading frame isdifferent relative to the corresponding wild-type sequence. In someembodiments, the uracil distribution within the open reading frame isdifferent relative to the corresponding wild-type sequence. In otherembodiments, the percentage of uracils of the total nucleotide contentin the open reading frame is unchanged relative to the correspondingwild-type sequence. In certain embodiments, the number of uracilclusters or the size of one or more uracil clusters in the open readingframe is different relative to the corresponding wild-type sequence. Inother embodiments, the distribution of uracil clusters in the openreading frame is different relative to the corresponding wild-typesequence. In certain embodiments, the distance between the uracilclusters or the location of one or more of the uracil clusters in theopen reading frame is different relative to the corresponding wild-typesequence.

In some embodiments, the mRNA does not contain more than fourconsecutive uracils.

In some embodiments, the uracil content of the open reading frame isbetween a theoretical minimum and 200% of the theoretical minimum (e.g.,between the theoretical minimum and 125% of the theoretical minimum,between the theoretical minimum and 150% of the theoretical minimum, orbetween the theoretical minimum and 175% of the theoretical minimum).

In other embodiments, the uracil content within any 20 nucleotide windowwithin the open reading frame does not exceed 50% (e.g., does not exceed40%, does not exceed 30%, does not exceed 20%, or does not exceed thetheorectical minimum).

In certain embodiments, the guanine content of the open reading frame ismaximized for at least 90% (e.g., at least 95%, at least 99%, or 100%)of the codons.

In some embodiments, the cytosine content of the open reading frame ismaximized for at least 90% (e.g., at least 95%, at least 99%, or 100%)of the codons.

In another aspect, the invention provides an mRNA encoding a polypeptideof interest and including an open reading frame, wherein (a) at least90% (e.g., at least 95%, at least 99%, or 100%) of the uracils in theopen reading frame are 5-methoxy-uracil and (b) at least 50% (e.g., atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%) of the codons in the open reading frame are guanineand/or cytosine maximized codons, wherein the open reading frameincludes at least one low frequency (i.e., a codon that is not thehighest frequency codon) guanine and/or cytosine maximized codon. Insome embodiments, at least 50% (e.g., at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 99%, or 100%) of thecodons in the open reading frame are guanine maximized codons, whereinthe open reading frame comprises at least one low frequency guaninemaximized codon. In other embodiments, at least 50% (e.g., at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or100%) of the codons in the open reading frame are cytosine maximizedcodons, wherein the open reading frame comprises at least one lowfrequency cytosine maximized codon. In certain embodiments, at least 50%(e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least95%, at least 99%, or 100%) of the codons in the open reading frame areguanine and cytosine maximized codons, wherein the open reading framecomprises at least one low frequency guanine and/or cytosine maximizedcodon.

In another aspect, the invention features an mRNA encoding a polypeptideof interest and including an open reading frame, wherein (a) at least90% (e.g., at least 95%, at least 99%, or 100%) of the uracils in theopen reading frame are 5-methoxy-uracil and (b) the open reading frameincludes at least one of the following codons: GCG, GGG, CCG, AGG, ACG,CUC, CGC, UCC, and GUC. In some embodiments, the open reading framecomprises at least one of the following codons: GCG, GGG, CCG, AGG, andACG. In other embodiments, the open reading frame comprises at least oneof the following codons: CUC, CGC, UCC, and GUC. In certain embodiments,the open reading frame comprises (i) at least one of the followingcodons: GCG, GGG, CCG, AGG, and ACG and (ii) at least one of thefollowing codons CUC, CGC, UCC, and GUC.

In other embodiments of any of the foregoing mRNAs, the sequence of themRNA has at least 55% (e.g., at least 60%, at least 65%, at least 70%,at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%)identity to the corresponding wild-type sequence. In some embodiments ofany of the foregoing mRNAs, the sequence of the mRNA has 60-80% (e.g.,65-75%, 60-65%, 65-70%, 70-75%, or 75-80%) identity to the correspondingwild-type sequence.

In certain embodiments of any of the foregoing mRNA, the mRNA furtherincludes:

-   -   (i) at least one 5′-cap structure;    -   (ii) a 5′-UTR; and    -   (iii) a ‘3’-UTR.

In some embodiments, the at least one 5′-cap structure is Cap0, Cap1,ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, or2-azido-guanosine.

In other embodiments of any of the foregoing mRNA, the mRNA furtherincludes a poly-A tail.

In certain embodiments of any of the foregoing mRNA, the mRNA ispurified.

In another aspect, the invention features a pharmaceutical compositionincluding any of the foregoing mRNA and a pharmaceutically acceptableexcipient.

In another aspect, the invention features any of the foregoing mRNA orpharmaceutical compositions, for use in therapy.

In some embodiments of any of the foregoing mRNA, the mRA induces adetectably lower innate immune response relative to the correspondingwild-type mRNA.

In other embodiments of any of the foregoing mRNA, the mRNA exhibitsenhanced ability to produce the encoded protein of interest in amammalian cell compared to the corresponding wild-type mRNA.

In some embodiments of any of the foregoing mRNA, the mRNA exhibitsincreased stability. For example, in some embodiments, the mRNA exhibitsincreased stability in a cell into which it is introduced, relative to acorresponding wild-type mRNA. In some embodiments of any of theforegoing mRNA, the mRNA exhibits increased stability includingresistance to nucleases, thermal stability, and/or increasedstabilization of secondary structure. In some embodiments of any of theforegoing mRNA, increased stability exhibited by the mRNA is measured bydetermining the half life of the mRNA (e.g., in a plasma, cell, ortissue sample) and/or determining the area under the curve (AUC) of theprotein expression by the mRNA over time (e.g., in vitro or in vivo). AnmRNA is identified as having increased stability if the half life and/orthe AUC is greater than the corresponding wild-type mRNA.

In some embodiments of any of the foregoing mRNA, the mRNA exhibitsenhanced ability to translate or to produce the encoded protein ofinterest, exhibits increased stability, and/or induces a detectablylower immune response (e.g., innate or acquired) relative to acorresponding wild-type mRNA and/or an mRNA including one or moredifferent alternative nucleic acids of the wild-type mRNA which havebeen altered in a different manner (e.g., an alternative nucleic acidincluding an alternative nucleosides other than 5-methoxy-uridine or analternative nucleic acid for which uridine content has not been reduced)in a cell such as in a mammalian cell.

In another aspect, the invention features a method of expressing apolypeptide of interest in a mammalian cell, the method including thesteps of:

(i) providing any of the foregoing mRNA; and

(ii) introducing the mRNA to a mammalian cell under conditions thatpermit the expression of the polypeptide of interest by the mammaliancell.

In another aspect, the invention features a composition including:

a) a DNA template;

b) an RNA polymerase;

c) ATP, GTP, CTP, and 5-methoxy-UTP; and

d) one or more copies of mRNA produced by the RNA polymerase, and

e) less than 70% (e.g., less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, less than 4%,less than 3%, less than 2%, less than 1%, between 0.01 and 5%, between1% and 10%, between 5% and 20%, between 10% and 30%, between 15% and40%, between 20% and 50%, between 30% and 60%, between 40% and 70%) ofaberrant transcription products relative to full length mRNA (e.g., asmeasured by moles of aberrant transcription products/moles of aberranttranscription products and moles full length mRNA).

In another aspect, the invention features the use of 5-methoxy-uridinein the production of a medicament including an mRNA, wherein theproduction does not include reverse phase purification, e.g. wherein thereverse phase purification is not needed to remove aberranttranscription products.

In another aspect, the invention features a method of producing apharmaceutical composition including mRNA molecules, the methodincluding:

a) performing in vitro synthesis to produce a composition including mRNAmolecules; and

b) determining the level of aberrant transcription products in thecomposition.

In another aspect, the invention features a method of producing apharmaceutical composition including mRNA molecules, the methodincluding:

a) performing in vitro transcription with an RNA polymerase and a DNAtemplate to produce a composition including mRNA molecules; and

b) determining the level of aberrant transcription products in thecomposition.

In some embodiments, the method further includes c) purifying thecomposition if the level of aberrant transcription products in thecomposition is greater than a predetermined level (e.g., 70%). In someembodiments, purifying includes reverse phase chromatography.

In another aspect, the invention features a method of producing mRNA,the method includes a purification step including removal of aberranttranscription products without reverse phase chromatography includingfor example affinity chromatography, precipitation, and/or membranepurification such as tangential flow filtration (TFF) methods as areknown in the art.

In another aspect, the invention features a method of producing apharmaceutical composition including mRNA, the method includingdetermining the level of mRNA including aberrant transcription productsin the composition.

In another aspect, the invention features a pharmaceutical compositionincluding mRNA, wherein the pharmaceutical composition has beendetermined to include less than less than 70% (e.g., less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, less than 10%,less than 5%, less than 4%, less than 3%, less than 2%, less than 1%,between 0.01 and 5%, between 1% and 10%, between 5% and 20%, between 10%and 30%, between 15% and 40%, between 20% and 50%, between 30% and 60%,between 40% and 70%) aberrant transcription products relative to fulllength mRNA (e.g., as measured by moles of aberrant transcriptionproducts/moles of aberrant transcription products and moles full lengthmRNA).

In some embodiments of the foregoing compositions, uses, and methods, acomposition including a lower amount of aberrant transcription productsexhibits decreased immunogenicity relative to a composition including ahigher amount of mRNA including aberrant transcription products. In someembodiments of the foregoing compositions, uses, and methods, the mRNAin the composition includes 5-methoxy-uridine.

In another aspect, the invention features a method of producing apharmaceutical composition including an mRNA comprising5-methoxy-uridine, the method including: (a) producing (e.g., directingthe production of) a composition including in vitro synthesized mRNAcomprising 5-methoxy-uridine; and (b) purifying (e.g., directing thepurification of) the composition without reverse phase chromatography,thereby producing a pharmaceutical composition including an mRNAcomprising 5-methoxy-uridine.

In some embodiments, step (a) includes in vitro transcription (e.g.,with ATP, GTP, CTP, and 5-methoxy-UTP, an RNA polymerase such as T7 RNApolymerase and a DNA template such as cDNA).

In some embodiments, the method further includes (c) formulating thecomposition for administration. In some embodiments, step (c) includesformulating the composition in unit dosage form. In some embodiments,formulating includes includes one or more of: processing the compositioninto a drug product; combining the composition with a second component,e.g., an excipient and/or diluent; changing the concentration of themRNA in the composition; lyophilizing the composition; combining a firstand second aliquot of the composition to provide a third, larger,aliquot; dividing the composition into smaller aliquots; disposing thecomposition into a container, e.g., a gas or liquid tight container;packaging the composition; and/or associating a container including thecomposition with a label (e.g., labeling).

In some embodiments, the invention features a method of producing apharmaceutical composition including an mRNA, wherein the mRNA comprises5-methoxy-uridine, the method including: (a) providing a compositionincluding in vitro synthesized mRNA; and (b) purifying (e.g., directingthe purification of) the composition without reverse phasechromatography, thereby producing a pharmaceutical composition includingmRNA.

In some embodiments, the in vitro synthesized mRNA is produced by invitro transcription including an RNA polymerase (e.g., T7 RNApolymerase) and a DNA template (e.g., cDNA).

In some embodiments, the method further includes (c) formulating thecomposition for administration. In some embodiments, step (c) includesformulating the composition in unit dosage form. In some embodiments,formulating includes includes one or more of: processing the compositioninto a drug product; combining the composition with a second component,e.g., an excipient or diluent; changing the concentration of the mRNA inthe composition; lyophilizing the composition; combining a first andsecond aliquot of the composition to provide a third, larger, aliquot;dividing the composition into smaller aliquots; disposing thecomposition into a container, e.g., a gas or liquid tight container;packaging the composition; and/or associating a container including thecomposition with a label (e.g., labeling).

In some embodiments, the composition exhibits decreased immunogenicityrelative to a composition including an mRNA that does not comprise5-methoxy-uridine and is produced by the same method. In someembodiments, the composition exhibits increased protein expressionrelative to a composition including an mRNA that does not comprise5-methoxy-uridine and is produced by the same method. In someembodiments, the mRNA comprising 5-methoxy-uridine exhibits increasedstability relative to an mRNA that does not comprise 5-methoxy-uridineand is produced by the same method. In some embodiments, the compositiondoes not exhibit decreased immunogenicity relative to a compositionincluding an mRNA that does not comprise 5-methoxy-uridine and isproduced by a method including reverse phase purification. In someembodiments, the composition includes fewer RNA impurities resultingfrom aberrant transcription products (e.g., short RNAs resulting fromabortive transcription, double stranded RNA resulting from RNA dependentRNA polymerase activity, and/or RNA including 3′ extension region)relative to a composition including an mRNA that does not comprise5-methoxy-uridine and is produced by the same method.

In another aspect, the invention features a pharmaceutical compositionincluding an mRNA comprising 5-methoxy-uridine produced by performing invitro transcription (e.g., with an RNA polymerase such as T7 RNApolymerase and a DNA template such as cDNA), to produce a compositionincluding the mRNA and purifying the composition without reverse phasechromatography.

In some embodiments, the composition is formulated for administration.In some embodiments, the composition is formulated in unit dosage form.In some embodiments, formulating the composition includes one or moreof: processing the composition into a drug product; combining thecomposition with a second component, e.g., an excipient or diluent;changing the concentration of the mRNA in the composition; lyophilizingthe composition; combining a first and second aliquot of the compositionto provide a third, larger, aliquot; dividing the composition intosmaller aliquots; disposing the composition into a container, e.g., agas or liquid tight container; packaging the composition; and/orassociating a container including the composition with a label (e.g.,labeling).

In some embodiments, the composition exhibits decreased immunogenicityrelative to a composition including mRNA that does not comprise5-methoxy-uridine produced with a method including purification withoutreverse phase chromatography. In some embodiments, the compositionexhibits increased protein expression relative to a compositionincluding mRNA that does not comprise 5-methoxy-uridine. In someembodiments, the mRNA exhibits increased stability relative to mRNA thatdoes not comprise 5-methoxy-uridine. In some embodiments, thecomposition does not exhibit decreased immunogenicity relative to acomposition including an mRNA that does not comprise 5-methoxy-uridineand that is purified by reverse phase chromatography. In someembodiments, the composition includes fewer RNA impurities such asaberrant transcription products (e.g., short RNAs resulting fromabortive transcription, double stranded RNA resulting from RNA dependentRNA polymerase activity, and/or RNA including a 3′ extension region)relative to a composition including an mRNA that does not comprise5-methoxy-uridine and is produced with a method including purificationwithout reverse phase chromatography.

In some embodiments of any of the foregoing methods or compositions, theaberrant transcription products include short RNAs as a result ofabortive transcription initiation events. In some embodiments of any ofthe foregoing methods or compositions, the aberrant transcriptionproducts include double stranded (ds)RNAs generated by RNA dependent RNApolymerase activity. In some embodiments of any of the foregoing methodsor compositions, the aberrant transcription products include RNA-primedtranscription from RNA templates. In some embodiments of any of theforegoing methods or compositions, the aberrant transcription productsinclude RNA comprising a self-complementary 3′ extension region.

In some embodiments of any of the foregoing methods or compositions, thelevel of RNA impurities and/or aberrant transcription products may bedetermined by any method known in the art (e.g., liquid chromatographysuch as HPLC, UPLC, or LC-MS analysis or capillary electrophoresis).

In some embodiments of any of the foregoing methods or compositions, themRNA comprises 5-methoxy-uridine. In some embodiments of any of theforegoing methods or compositions at least 5% (e.g., at least 10%, atleast 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 99%, or 100%) of the uridines in the mRNA are 5-methoxy-uridine.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of protein expression by uridine-minimized mRNArelative to the corresponding wild-type mRNA.

FIG. 2 is a graph of protein expression by uridine-minimized mRNArelative to the corresponding wild-type mRNA.

FIG. 3 is a graph of the induction of INFβ by mRNA produced by IVT atdifferent temperatures.

FIG. 4 is a graph of the induction of INFβ by mRNA with 3′-terminalpoly-U feature.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides, inter alia, alternative nucleosides,alternative nucleotides, and alternative nucleic acids that exhibitimproved therapeutic properties including, but not limited to, increasedexpression and/or a reduced innate immune response when introduced intoa population of cells.

As there remains a need in the art for therapeutic modalities to addressthe myriad barriers surrounding the efficacious modulation ofintracellular translation and processing of nucleic acids encodingpolypeptides or fragments thereof, the inventors have shown that certainalternative mRNA sequences have the potential as therapeutics withbenefits beyond just evading, avoiding or diminishing the immuneresponse.

The present invention addresses this need by providing nucleic acidbased compounds or polynucleotides (e.g., alternative mRNAs) whichencode a polypeptide of interest and which have structural and/orchemical features that avoid one or more of the problems in the art, forexample, features which are useful for optimizing nucleic acid-basedtherapeutics while retaining structural and functional integrity,overcoming the threshold of expression, improving expression rates, halflife and/or protein concentrations, optimizing protein localization, andavoiding deleterious bio-responses such as the immune response and/ordegradation pathways.

In particular, the inventors have identified that mRNA wherein theuracil content has been modified may be particularly effective for usein therapeutic compositions, because they may benefit from both highexpression levels and limited induction of the innate immune response,as shown in the Examples (in particular, high performance may beobserved across the assays in Examples 6-9). In the invention, apercentage of the uracils in the open reading frame (and optionallyother components of an mRNA) are 5-methoxy-uracil. Preferaby, at least90%, e.g., at least 95% or 100%, of the uracils are 5-methoxy-uracil.Thus, as is apparent from the context, the term uracils can refer to5-methoxy-uracil and naturally occurring uracil.

Polypeptides of interest, according to the present invention, may beselected from any of those disclosed in US 2013/0259924, US2013/0259923, WO 2013/151663, WO 2013/151669, WO 2013/151670, WO2013/151664, WO 2013/151665, WO 2013/151736, U.S. Provisional PatentApplication No. 61/618,862, U.S. Provisional Patent Application No.61/681,645, U.S. Provisional Patent Application No. 61/618,873, U.S.Provisional Patent Application No. 61/681,650, U.S. Provisional PatentApplication No. 61/618,878, U.S. Provisional Patent Application No.61/681,654, U.S. Provisional Patent Application No. 61/618,885, U.S.Provisional Patent Application No. 61/681,658, U.S. Provisional PatentApplication No. 61/618,911, U.S. Provisional Patent Application No.61/681,667, U.S. Provisional Patent Application No. 61/618,922, U.S.Provisional Patent Application No. 61/681,675, U.S. Provisional PatentApplication No. 61/618,935, U.S. Provisional Patent Application No.61/681,687, U.S. Provisional Patent Application No. 61/618,945, U.S.Provisional Patent Application No. 61/681,696, U.S. Provisional PatentApplication No. 61/618,953, and U.S. Provisional Patent Application No.61/681,704, the polypeptides of each of which are incorporated herein byreference.

Provided herein, in part, are polynucleotides encoding polypeptides ofinterest which have been chemically modified to improve one or more ofthe stability and/or clearance in tissues, receptor uptake and/orkinetics, cellular access by the compositions, engagement withtranslational machinery, mRNA half-life, translation efficiency, immuneevasion, protein production capacity, secretion efficiency (whenapplicable), accessibility to circulation, protein half-life and/ormodulation of a cell's status, function and/or activity.

The alternative polynucleotides of the invention, including thecombination of alterations taught herein, have superior propertiesmaking them more suitable as therapeutic modalities.

In one aspect of the invention, methods of determining the effectivenessof an alternative mRNA as compared to wild-type involves the measure andanalysis of one or more cytokines whose expression is triggered by theadministration of the exogenous nucleic acid of the invention. Thesevalues are compared to administration of an unaltered nucleic acid or toa standard metric such as cytokine response, PolyIC, R-848 or otherstandard known in the art.

One example of a standard metric herein is the measure of the ratio ofthe level or amount of encoded polypeptide (protein) produced in thecell, tissue or organism to the level or amount of one or more (or apanel) of cytokines whose expression is triggered in the cell, tissue ororganism as a result of administration or contact with the alternativenucleic acid. Such ratios are referred to herein as the Protein:CytokineRatio or “PC” Ratio. The higher the PC ratio, the more efficacious thealternative nucleic acid (polynucleotide encoding the protein measured).Preferred PC Ratios, by cytokine, of the present invention may begreater than 1, greater than 10, greater than 100, greater than 1000,greater than 10,000 or more. Alternative nucleic acids having higher PCRatios than an alternative nucleic acid of a different or unalteredconstruct are preferred.

The PC ratio may be further qualified by the percent alteration presentin the polynucleotide. For example, normalized to a 100% alternativenucleic acid, the protein production as a function of cytokine (or risk)or cytokine profile can be determined.

Preferably, the alternative mRNAs are substantially non toxic and nonmutagenic.

The compositions and methods described herein can be used, in vivo andin vitro, both extracellularly and intracellularly, as well as in assayssuch as cell free assays.

In another aspect, the present disclosure provides chemical alterationslocated on the sugar moiety of the nucleotide.

In another aspect, the present disclosure provides chemical alterationslocated on the phosphate backbone of the nucleic acid.

In another aspect, the present disclosure provides nucleotides thatcontain chemical alterations, wherein the nucleotide reduces thecellular innate immune response, as compared to the cellular innateimmune induced by a corresponding unaltered nucleic acid.

In another aspect, the present disclosure provides compositionscomprising a compound as described herein. In some embodiments, thecomposition is a reaction mixture. In some embodiments, the compositionis a pharmaceutical composition. In some embodiments, the composition isa cell culture. In some embodiments, the composition further comprisesan RNA polymerase and a cDNA template. In some embodiments, thecomposition further comprises a nucleotide having a nucleobase selectedfrom the group consisting of adenine, cytosine, guanine,5-methoxy-uracil and/or uracil.

In a further aspect, the present disclosure provides methods of making apharmaceutical formulation comprising a physiologically active secretedprotein, comprising transfecting a first population of human cells withthe pharmaceutical nucleic acid made by the methods described herein,wherein the secreted protein is active upon a second population of humancells.

In some embodiments, the secreted protein is capable of interacting witha receptor on the surface of at least one cell present in the secondpopulation.

In certain embodiments, provided herein are combination therapeuticscontaining one or more alternative nucleic acids containing translatableregions that encode for a protein or proteins that boost a mammaliansubject's immunity along with a protein that induces antibody-dependentcellular toxicity.

In one embodiment, it is intended that the compounds of the presentdisclosure are stable. It is further appreciated that certain featuresof the present disclosure, which are, for clarity, described in thecontext of separate embodiments, can also be provided in combination ina single embodiment. Conversely, various features of the presentdisclosure which are, for brevity, described in the context of a singleembodiment, can also be provided separately or in any suitablesubcombination.

Uracil Content

The present disclosure provides nucleic acids wherein with altereduracil content at least one codon in the wild-type sequence has beenreplaced with an alternative codon to generate a uracil-alteredsequence. Altered uracil sequences can have at least one of thefollowing properties:

-   -   (i) an increase or decrease in global uracil content (i.e., the        percentage of uracil of the total nucleotide content in the        nucleic acid of a section of the nucleic acid, e.g., the open        reading frame); or,    -   (ii) an increase or decrease in local uracil content (i.e.,        changes in uracil content are limited to specific subsequences);        or,    -   (iii) a change in uracil distribution without a change in the        global uracil content; or,    -   (iv) a change in uracil clustering (e.g., number of clusters,        location of clusters, or distance between clusters); or,    -   (v) combinations thereof.

In some aspects, the percentage of uracil nucleobases in the nucleicacid sequence is reduced with respect to the percentage of uracilnucleobases in the wild-type nucleic acid sequence. For example, 30% ofnucleobases may be uracils in the wild-type sequence and 10% in thenucleic acid sequence of the invention. The percentage uracil contentcan be determined by dividing the number of uracils in a sequence by thetotal number of nucleotides and multiplying by 100.

In other aspects, the percentage of uracil nucleobases in a subsequenceof the nucleic acid sequence is reduced with respect to the percentageof uracil nucleobases in the corresponding subsequence of the wild-typesequence. For example, the wild-type sequence may have a 5′-end region(e.g., 30 codons) with a local uracil content of 30%, and the uracilcontent in that same region could be reduced to 10% in the nucleic acidsequence of the invention.

In specific aspects, codons in the nucleic acid sequence of theinvention reduce or modify, for example, the number, size, location, ordistribution of uracil clusters that could have deleterious effects onprotein translation. Although as a general rule lower uracil content isdesirable, in certain aspects, the uracil content, and in particular thelocal uracil content, of some subsequences of the wild-type sequence canbe greater than the wild-type sequence and still maintain beneficialfeatures (e.g., increased expression).

In some aspects, the uracil-modified sequence induces a lower Toll-LikeReceptor (TLR) response when compared to the wild-type sequence. SeveralTLRs recognize and respond to nucleic acids. Double-stranded (ds)RNA, afrequent viral constituent, has been shown to activate TLR3. SeeAlexopoulou et al. (2001) Nature, 413:732-738 and Wang et al. (2004)Nat. Med., 10:1366-1373. Single-stranded (ss)RNA activates TLR7. SeeDiebold et al. (2004) Science 303:1529-1531. RNA oligonucleotides, forexample RNA with phosphorothioate internucleotide linkages, are ligandsof human TLR8. See Heil et al. (2004) Science 303:1526-1529. DNAcontaining unmethylated CpG motifs, characteristic of bacterial andviral DNA, activate TLR9. See Hemmi et al. (2000) Nature, 408: 740-745.See also, Kariko et al. (2005) Immunity 23:165-175, which is hereinincorporated by reference in its entirety.

As used herein, the term “TLR response” is defined as the recognition ofsingle-stranded RNA by a TLR7 receptor, and in some aspects encompassesthe degradation of the RNA and/or physiological responses caused by therecognition of the single-stranded RNA by the receptor. Methods todetermine and quantify the binding of an RNA to a TLR7 are known in theart. Similarly, methods to determine whether an RNA has triggered aTLR7-mediated physiological response (e.g., cytokine secretion) are wellknown in the art. In some aspects, a TLR response can be mediated byTLR3, TLR8, or TLR9 instead of TLR7.

Suppression of TLR7-mediated response can be accomplished via nucleosidemodification. RNA undergoes over a hundred different nucleosidemodifications in nature (see the RNA Modification Database, available atmods.rna.albany.edu). Human rRNA, for example, has ten times morepseudouracil (ψ) and 25 times more 2′-O-methylated nucleosides thanbacterial rRNA. Bacterial mRNA contains no nucleoside modifications,whereas mammalian mRNAs have modified nucleosides such as5-methylcytidine (m5C), N6-methyladenosine (m6A), inosine and many2′-O-methylated nucleosides in addition to N7-methylguanosine (m7G).

Uracil and ribose, the two defining features of RNA, are both necessaryand sufficient for TLR7 stimulation, and short single-stranded RNA(ssRNA) act as TLR7 agonists in a sequence-independent manner as long asthey contain several uracils in close proximity. See Diebold et al.(2006) Eur. J. Immunol. 36:3256-3267, which is herein incorporated byreference in its entirety. Accordingly, a nucleic acid sequence of theinvention may have reduced uracil content (locally and/or locally)and/or reduced or altered uracil clustering to reduce or to suppress aTLR7-mediated response.

In some aspects, the TLR response (e.g., a response mediated by TLR7)caused by the uracil-modified sequence is at least about 10%, at leastabout 15%, at least about 20%, at least about 25%, at least about 30%,at least about 35%, at least about 40%, at least about 45%, at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, or at least 100%lower than the TLR response caused by the wild-type nucleic acidsequence.

In some aspects, the TLR response caused by the wild-type nucleic acidis at least about 1-fold, at least about 1.1-fold, at least about1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at leastabout 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, atleast about 1.8-fold, at least about 1.9-fold, at least about 2-fold, atleast about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 6-fold, at least about 7-fold, at least about 8-fold, atleast about 9-fold, or at least about 10-fold higher than the TLRresponse caused by the uracil-modified sequence.

In other aspects, the uracil content of the uracil-altered sequence islower than the uracil content of the wild-type nucleic acid sequence.Accordingly, in some aspects, the sequence of the invention contains atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 100% less uracil than thewild-type nucleic acid sequence.

In some aspects, the uracil content is less than 50%, 49%, 48%, 47%,46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%,32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% 5%, 4%, 3%,2% or 1% of the total nucleobases in the sequence of the invention. Insome aspects, the uracil content of the sequence of the invention isbetween about 5% and about 25%. In some particular aspects, the uracilcontent of the sequence of the invention is between about 15% and about25%.

In some aspects, the uracil content of the wild-type nucleic acidsequence can be measured using a sliding window. In some aspects, thelength of the sliding window is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, or 40 nucleobases. In some aspects, the slidingwindow is over 40 nucleobases in length. In a preferred aspect, thesliding window is 20 nucleobases in length. Based on the uracil contentmeasured with a sliding window, it is possible to generate a histogramrepresenting the uracil content throughout the length of the wild-typenucleic acid sequence and nucleic acid sequence of the invention. Insome aspects, the nucleic acid sequence of the invention has fewer peaksin the representation that are above a certain percentage value relativeto the candidate sequence. In some aspects, the nucleic acid sequence ofthe invention does not have peaks in the sliding-window representationwhich are above 65%, 60%, 55%, 50%, 45%, 40%, 35%, or 30% uracil. In apreferred aspect, the nucleic acid sequence of the invention has nopeaks over 30% uracil, as measured using a 20 nucleobase sliding window.In some aspects, the nucleic acid sequence of the invention has no morethan a predetermined number of peaks, as measured using a 20 nucleobasesliding window, above a certain threshold value. For example, in someaspects, the nucleic acid sequence of the invention has no peaks or nomore than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 peaks in the above 10%, 15%,20%, 25% or 30% uracil. In a preferred aspect, the nucleic acid sequenceof the invention contains between 0 peaks and 2 peaks with uracilcontent 30% or higher.

In some aspects, the nucleic acid sequence has reduced consecutiveuracils. For example, two consecutive leucines could be encoded by thesequence CUUUUG, which would include a four uracil cluster. Such asubsequence could be substituted with CUGCUC, which would effectivelyremove the uracil cluster. Accordingly, a nucleic sequence may havereduced or no uracil pairs (UU), uracil triplets (UUU) or uracilquadruplets (UUUU), relative to the wild-type nucleic acid sequence. Insome aspects, the nucleic acid sequence does not include uracil pairs(UU) and/or uracil triplets (UUU) and/or uracil quadruplets (UUUU). Inother aspects, the nucleic acid sequence does not include uracil pairs(UU) and/or uracil triplets (UUU) and/or uracil quadruplets (UUUU) abovea certain threshold, e.g., no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10occurrences in the nucleic acid sequence. In a particular aspect, thenucleic acid sequence contains fewer than 5, 4, 3, 2, or 1 uracil pairs.In another particular aspect, the nucleic acid sequence contains nouracil pairs.

In some aspects, the wild-type nucleic acid sequence can comprise uracilclusters which due to their number, size, location, distribution orcombinations thereof have negative effects on translation. As usedherein, the term “uracil cluster” refers to a subsequence in a nucleicacid sequence that contains a uracil content (usually described as apercentage) which is above a certain threshold. Thus, in certainaspects, if a subsequence comprises more than 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60% or 65% uracil content, such subsequencewould be considered a uracil cluster.

The negative effects of uracil clusters can be, for example, eliciting aTLR7 response. Thus, in some embodiments, the nucleic acid of theinvention has a reduced number of clusters, size of clusters, locationof clusters (e.g., close to the 5′ and/or 3′ end of a nucleic acidsequence), distance between clusters, or distribution of uracil clusters(e.g., a certain pattern of clusters along a nucleic acid sequence,distribution of clusters with respect to secondary structure elements inthe expressed product, or distribution of clusters with respect to thesecondary structure of an mRNA) relative to wild-type.

In some aspects, the wild-type sequence comprises at least one uracilcluster, wherein said uracil cluster is a subsequence of the wild-typenucleic acid sequence wherein the percentage of total uracil nucleobasesin said subsequence is above a predetermined threshold. In some aspects,the length of the subsequence is at least about 10, at least about 15,at least about 20, at least about 25, at least about 30, at least about35, at least about 40, at least about 45, at least about 50, at leastabout 55, at least about 60, at least about 65, at least about 70, atleast about 75, at least about 80, at least about 85, at least about 90,at least about 95, or at least about 100 nucleobases. In some aspects,the subsequence is longer than 100 nucleobases. In some aspects, thethreshold is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% or 25% uracilcontent. In some aspects, the threshold is above 25%.

For example, an amino acid sequence such as ADGSR could be encoded bythe nucleic acid sequence GCU GAU GGU AGU CGU. Although such a sequencedoes not contain any uracil pairs, triplets, or quadruplets, one thirdof the nucleobases would be uracils. Such a uracil cluster could beremoved by using alternative codons, for example, by using the codingsequence GCC GAC GGC AGC CGC, which would contain no uracils.

In other aspects, the wild-type sequence comprises at least one uracilcluster, wherein said uracil cluster is a subsequence of the wild-typenucleic acid sequence wherein the percentage of uracil nucleobases ofsaid subsequence as measured using a sliding window is above apredetermined threshold. In some aspects, the length of the slidingwindow is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,or 40 nucleobases. In some aspects, the sliding window is over 40nucleobases in length. In some aspects, the threshold is 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, 21%, 22%, 23%, 24% or 25% uracil content. In some aspects, thethreshold is above 25%.

In some aspects, the wild-type nucleic acid sequence comprises at leasttwo uracil clusters. In some aspects, the sequence of the inventioncontains fewer uracil-rich clusters than the wild-type nucleic acidsequence. In some aspects, the sequence of the invention contains moreuracil-rich clusters than the wild-type nucleic acid sequence. In someaspects, the sequence of the invention contains uracil-rich clusterswhich are shorter in length than corresponding uracil-rich clusters inthe wilde type nucleic acid sequence. In other aspects, the sequence ofthe invention contains uracil-rich clusters which are longer in lengththat corresponding uracil-rich cluster in the wild-type nucleic acidsequence.

Alternative Nucleotides, Nucleosides and Polynucleotides of theInvention

Herein, in a nucleotide, nucleoside or polynucleotide (such as thenucleic acids of the invention, e.g., mRNA molecule), the terms“alteration” or, as appropriate, “alternative” refer to alteration withrespect to A, G, U or C ribonucleotides. Generally, herein, these termsare not intended to refer to the ribonucleotide alterations in naturallyoccurring 5′-terminal mRNA cap moieties. In a polypeptide, the term“alteration” refers to an alteration as compared to the canonical set of20 amino acids, moiety)

The alterations may be various distinct alterations. In someembodiments, where the nucleic acid is an mRNA, the coding region, theflanking regions and/or the terminal regions may contain one, two, ormore (optionally different) nucleoside or nucleotide alterations. Insome embodiments, an alternative polynucleotide introduced to a cell mayexhibit reduced degradation in the cell, as compared to an unalteredpolynucleotide.

The polynucleotides can include any useful alteration, such as to thesugar or the internucleoside linkage (e.g., to a linking phosphate/to aphosphodiester linkage/to the phosphodiester backbone). In certainembodiments, alterations (e.g., one or more alterations) are present ineach of the sugar and the internucleoside linkage. Alterations accordingto the present invention may be alterations of ribonucleic acids (RNAs)to deoxyribonucleic acids (DNAs), e.g., the substitution of the 2′OH ofthe ribofuranosyl ring to 2′H, threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs) or hybrids thereof). Additional alterations are described herein.

As described herein, the polynucleotides of the invention do notsubstantially induce an innate immune response of a cell into which thepolynucleotide (e.g., mRNA) is introduced. Features of an induced innateimmune response include 1) increased expression of pro-inflammatorycytokines, 2) activation of intracellular PRRs (RIG-I, MDAS, etc),and/or 3) termination or reduction in protein translation.

In certain embodiments, it may desirable for an alternative nucleic acidmolecule introduced into the cell to be degraded intracellularly. Forexample, degradation of an alternative nucleic acid molecule may bepreferable if precise timing of protein production is desired. Thus, insome embodiments, the invention provides an alternative nucleic acidmolecule containing a degradation domain, which is capable of beingacted on in a directed manner within a cell.

The polynucleotides can optionally include other agents (e.g.,RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisenseRNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helixformation, aptamers, vectors, etc.). In some embodiments, thepolynucleotides may include one or more messenger RNAs (mRNAs) havingone or more alternative nucleoside or nucleotides (i.e., alternativemRNA molecules). Details for these polynucleotides follow.

Polynucleotides

The polynucleotides of the invention typically include a first region oflinked nucleosides encoding a polypeptide of interest, a first flankingregion located at the 5′ terminus of the first region, and a secondflanking region located at the 3′ terminus of the first region.

Alterations on the Sugar

The alternative nucleosides and nucleotides, which may be incorporatedinto a polynucleotide (e.g., RNA or mRNA, as described herein), can bealtered on the sugar of the ribonucleic acid. For example, the 2′hydroxyl group (OH) can be modified or replaced with a number ofdifferent substituents. Exemplary substitutions at the 2′-positioninclude, but are not limited to, H, halo, optionally substituted C₁₋₆alkyl; optionally substituted C₁₋₆ alkoxy; optionally substituted C₆₋₁₀aryloxy; optionally substituted C₃₋₈ cycloalkyl; optionally substitutedC₃₋₈ cycloalkoxy; optionally substituted C₆₋₁₀ aryloxy; optionallysubstituted C₆₋₁₀ aryl-C₁₋₆ alkoxy, optionally substituted C₁₋₁₂(heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any describedherein); a polyethyleneglycol (PEG), —O(CH₂CH₂O)_(n)CH₂CH₂OR, where R isH or optionally substituted alkyl, and n is an integer from 0 to 20(e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4,from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from4 to 10, from 4 to 16, and from 4 to 20); “locked” nucleic acids (LNA)in which the 2′-hydroxyl is connected by a C₁₋₆ alkylene or C₁₋₆heteroalkylene bridge to the 4′-carbon of the same ribose sugar, whereexemplary bridges included methylene, propylene, ether, or aminobridges; aminoalkyl; aminoalkoxy; amino; and amino acid.

Generally, RNA includes the sugar group ribose, which is a 5-memberedring having an oxygen. Exemplary, non-limiting alternative nucleotidesinclude replacement of the oxygen in ribose (e.g., with S, Se, oralkylene, such as methylene or ethylene); addition of a double bond(e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ringcontraction of ribose (e.g., to form a 4-membered ring of cyclobutane oroxetane); ring expansion of ribose (e.g., to form a 6- or 7-memberedring having an additional carbon or heteroatom, such as anhydrohexitol,altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that alsohas a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and“unlocked” forms, such as glycol nucleic acid (GNA) (e.g., R-GNA orS-GNA, where ribose is replaced by glycol units attached tophosphodiester bonds), threose nucleic acid (TNA, where ribose isreplace with α-L-threofuranosyl-(3′→2′)), and peptide nucleic acid (PNA,where 2-amino-ethyl-glycine linkages replace the ribose andphosphodiester backbone). The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a polynucleotide moleculecan include nucleotides containing, e.g., arabinose, as the sugar.

Alterations on the Internucleoside Linkage

The alternative nucleotides, which may be incorporated into apolynucleotide molecule, can be altered on the internucleoside linkage(e.g., phosphate backbone). Herein, in the context of the polynucleotidebackbone, the phrases “phosphate” and “phosphodiester” are usedinterchangeably. Backbone phosphate groups can be altered by replacingone or more of the oxygen atoms with a different substituent.

The alternative nucleosides and nucleotides can include the wholesalereplacement of an unaltered phosphate moiety with anotherinternucleoside linkage as described herein. Examples of alternativephosphate groups include, but are not limited to, phosphorothioate,phosphoroselenates, boranophosphates, boranophosphate esters, hydrogenphosphonates, phosphoramidates, phosphorodiamidates, alkyl or arylphosphonates, and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bealtered by the replacement of a linking oxygen with nitrogen (bridgedphosphoramidates), sulfur (bridged phosphorothioates), and carbon(bridged methylene-phosphonates).

The alternative nucleosides and nucleotides can include the replacementof one or more of the non-bridging oxygens with a borane moiety (BH₃),sulfur (thio), methyl, ethyl and/or methoxy. As a non-limiting example,two non-bridging oxygens at the same position (e.g., the alpha (α), beta(β) or gamma (γ) position) can be replaced with a sulfur (thio) and amethoxy.

The replacement of one or more of the oxygen atoms at the a position ofthe phosphate moiety (e.g., α-thio phosphate) is provided to conferstability (such as against exonucleases and endonucleases) to RNA andDNA through the unnatural phosphorothioate backbone linkages.Phosphorothioate DNA and RNA have increased nuclease resistance andsubsequently a longer half-life in a cellular environment. While notwishing to be bound by theory, phosphorothioate linked polynucleotidemolecules are expected to also reduce the innate immune response throughweaker binding/activation of cellular innate immune molecules.

Other internucleoside linkages that may be employed according to thepresent invention, including internucleoside linkages which do notcontain a phosphorous atom, are described herein below.

Synthesis of Polynucleotide Molecules

The polynucleotide molecules for use in accordance with the inventionmay be prepared according to any useful technique, as described herein.The alternative nucleosides and nucleotides used in the synthesis ofpolynucleotide molecules disclosed herein can be prepared from readilyavailable starting materials using the following general methods andprocedures. Where typical or preferred process conditions (e.g.,reaction temperatures, times, mole ratios of reactants, solvents,pressures, etc.) are provided, a skilled artisan would be able tooptimize and develop additional process conditions. Optimum reactionconditions may vary with the particular reactants or solvent used, butsuch conditions can be determined by one skilled in the art by routineoptimization procedures.

The processes described herein can be monitored according to anysuitable method known in the art. For example, product formation can bemonitored by spectroscopic means, such as nuclear magnetic resonancespectroscopy (e.g., ¹H or ¹³C) infrared spectroscopy, spectrophotometry(e.g., UV-visible), or mass spectrometry, or by chromatography such ashigh performance liquid chromatography (HPLC) or thin layerchromatography.

Preparation of polynucleotide molecules of the present invention caninvolve the protection and deprotection of various chemical groups. Theneed for protection and deprotection, and the selection of appropriateprotecting groups can be readily determined by one skilled in the art.The chemistry of protecting groups can be found, for example, in Greene,et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons,1991, which is incorporated herein by reference in its entirety.

The reactions of the processes described herein can be carried out insuitable solvents, which can be readily selected by one of skill in theart of organic synthesis. Suitable solvents can be substantiallynonreactive with the starting materials (reactants), the intermediates,or products at the temperatures at which the reactions are carried out,i.e., temperatures which can range from the solvent's freezingtemperature to the solvent's boiling temperature. A given reaction canbe carried out in one solvent or a mixture of more than one solvent.Depending on the particular reaction step, suitable solvents for aparticular reaction step can be selected.

Resolution of racemic mixtures of alternative polynucleotides or nucleicacids (e.g., polynucleotides or alternative mRNA molecules) can becarried out by any of numerous methods known in the art. An examplemethod includes fractional recrystallization using a “chiral resolvingacid” which is an optically active, salt-forming organic acid. Suitableresolving agents for fractional recrystallization methods are, forexample, optically active acids, such as the D and L forms of tartaricacid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid,malic acid, lactic acid or the various optically active camphorsulfonicacids. Resolution of racemic mixtures can also be carried out by elutionon a column packed with an optically active resolving agent (e.g.,dinitrobenzoylphenylglycine). Suitable elution solvent composition canbe determined by one skilled in the art.

Alternative nucleosides and nucleotides (e.g., building block molecules)can be prepared according to the synthetic methods described in Ogata etal., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res.22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568(1962); and Xu et al., Tetrahedron, 48(9): 1729-1740 (1992), each ofwhich are incorporated by reference in their entirety.

The polynucleotides of the invention may or may not be uniformly alteredalong the entire length of the molecule. For example, one or more or alltypes of nucleotide (e.g., purine or pyrimidine, or any one or more orall of A, G, U, C) may or may not be uniformly altered in apolynucleotide of the invention, or in a given predetermined sequenceregion thereof. In some embodiments, all nucleotides X in apolynucleotide of the invention (or in a given sequence region thereof)are altered, wherein X may any one of nucleotides A, G, U, C, or any oneof the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C orA+G+C.

Different sugar alterations and/or internucleoside linkages (e.g.,backbone structures) may exist at various positions in thepolynucleotide. One of ordinary skill in the art will appreciate thatthe nucleotide analogs or other alteration(s) may be located at anyposition(s) of a polynucleotide such that the function of thepolynucleotide is not substantially decreased. An alteration may also bea 5′ or 3′ terminal alteration. The polynucleotide may contain fromabout 1% to about 100% alternative nucleotides (either in relation tooverall nucleotide content, or in relation to one or more types ofnucleotide, i.e., any one or more of A, G, U or C) or any interveningpercentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%,from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20%to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95%to 100%). It will be understood that any remaining percentage isaccounted for by the presence of A, G, U, or C.

Alternative Nucleic Acids

The present disclosure provides nucleic acids (or polynucleotides),including RNAs such as mRNAs that contain one or more alternativenucleosides (termed “alternative nucleic acids”) or nucleotides asdescribed herein, which have useful properties including the lack of asubstantial induction of the innate immune response of a cell into whichthe mRNA is introduced. Because these alternative nucleic acids enhancethe efficiency of protein production, intracellular retention of nucleicacids, and viability of contacted cells, as well as possess reducedimmunogenicity, these nucleic acids having these properties are alsotermed “enhanced nucleic acids” herein.

The term “nucleic acid,” in its broadest sense, includes any compoundand/or substance that is or can be incorporated into an oligonucleotidechain. In this context, the term nucleic acid is used synonymously withpolynucleotide. Exemplary nucleic acids for use in accordance with thepresent disclosure include, but are not limited to, one or more of DNA,RNA including messenger mRNA (mRNA), hybrids thereof, RNAi-inducingagents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes,catalytic DNA, RNAs that induce triple helix formation, aptamers, andvectors.

Provided are alternative nucleic acids containing a translatable regionand one, two, or more than two different nucleoside alterations. In someembodiments, the alternative nucleic acid exhibits reduced degradationin a cell into which the nucleic acid is introduced, relative to acorresponding unaltered nucleic acid. Exemplary nucleic acids includeribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleicacids (TNAs), glycol nucleic acids (GNAs), or a hybrid thereof. Inpreferred embodiments, the alternative nucleic acid includes messengerRNAs (mRNAs). As described herein, the nucleic acids of the presentdisclosure do not substantially induce an innate immune response of acell into which the mRNA is introduced.

In certain embodiments, it is desirable to intracellularly degrade analternative nucleic acid introduced into the cell, for example ifprecise timing of protein production is desired. Thus, the presentdisclosure provides an alternative nucleic acid containing a degradationdomain, which is capable of being acted on in a directed manner within acell.

Other components of nucleic acid are optional, and are beneficial insome embodiments. For example, a 5′ untranslated region (UTR) and/or a3′-UTR are provided, wherein either or both may independently containone or more different nucleoside alterations. In such embodiments,nucleoside alterations may also be present in the translatable region.Also provided are nucleic acids containing a Kozak sequence.

Additionally, provided are nucleic acids containing one or more intronicnucleotide sequences capable of being excised from the nucleic acid.

Further, provided are nucleic acids containing an internal ribosomeentry site (IRES). An IRES may act as the sole ribosome binding site, ormay serve as one of multiple ribosome binding sites of an mRNA. An mRNAcontaining more than one functional ribosome binding site may encodeseveral peptides or polypeptides that are translated independently bythe ribosomes (“multicistronic mRNA”). When nucleic acids are providedwith an IRES, further optionally provided is a second translatableregion. Examples of IRES sequences that can be used according to thepresent disclosure include without limitation, those from picornaviruses(e.g. FMDV), pest viruses (CFFV), polio viruses (PV),encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),murine leukemia virus (MLV), simian immune deficiency viruses (SIV) orcricket paralysis viruses (CrPV).

Major Groove Interacting Partners

As described herein, the phrase “major groove interacting partner”refers to RNA recognition receptors that detect and respond to RNAligands through interactions, e.g. binding, with the major groove faceof a nucleotide or nucleic acid. As such, RNA ligands comprisingalternative nucleotides or nucleic acids as described herein decreaseinteractions with major groove binding partners, and therefore decreasean innate immune response, or expression and secretion ofpro-inflammatory cytokines, or both.

Example major groove interacting, e.g. binding, partners include, butare not limited to the following nucleases and helicases. Withinmembranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single-and double-stranded RNAs. Within the cytoplasm, members of thesuperfamily 2 class of DEX(D/H) helicases and ATPases can sense RNAs toinitiate antiviral responses. These helicases include the RIG-I(retinoic acid-inducible gene I) and MDAS (melanomadifferentiation-associated gene 5). Other examples include laboratory ofgenetics and physiology 2 (LGP2), HIN-200 domain containing proteins, orHelicase-domain containing proteins.

Prevention or Reduction of Innate Cellular Immune Response

The term “innate immune response” includes a cellular response toexogenous single stranded nucleic acids, generally of viral or bacterialorigin, which involves the induction of cytokine expression and release,particularly the interferons, and cell death. Protein synthesis is alsoreduced during the innate cellular immune response. While it isadvantageous to eliminate the innate immune response triggered byintroduction of exogenous nucleic acids in a cell, the presentdisclosure provides alternative nucleic acids such as mRNAs thatsubstantially reduce the immune response, including interferonsignaling, without entirely eliminating such a response. In someembodiments, the immune response is reduced by 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as comparedto the immune response induced by a corresponding unaltered nucleicacid. Such a reduction can be measured by expression or activity levelof Type 1 interferons or the expression of interferon-regulated genessuch as the toll-like receptors (e.g., TLR7 and TLR8). Reduction or lackof induction of innate immune response can also be measured by decreasedcell death following one or more administrations of alternative RNAs toa cell population; e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%,95%, or over 95% less than the cell death frequency observed with acorresponding unaltered nucleic acid. Moreover, cell death may affectfewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than0.01% of cells contacted with the alternative nucleic acids.

In some embodiments, the alternative nucleic acids, includingpolynucleotides and/or mRNA molecules are alternative in such a way asto not induce, or induce only minimally, an immune response by therecipient cell or organism. Such evasion or avoidance of an immuneresponse trigger or activation is a novel feature of the alternativepolynucleotides of the present invention.

The present disclosure provides for the repeated introduction (e.g.,transfection) of alternative nucleic acids into a target cellpopulation, e.g., in vitro, ex vivo, or in vivo. The step of contactingthe cell population may be repeated one or more times (such as two,three, four, five or more than five times). In some embodiments, thestep of contacting the cell population with the alternative nucleicacids is repeated a number of times sufficient such that a predeterminedefficiency of protein translation in the cell population is achieved.Given the reduced cytotoxicity of the target cell population provided bythe nucleic acid alterations, such repeated transfections are achievablein a diverse array of cell types in vitro and/or in vivo.

Minimization of RNA Impurities to Decrease Innate Immune Response

In some embodiments, RNA impurities (e.g., aberrant transcriptionproducts) in a composition including mRNA induce an immune response. TheRNA impurities such as, short RNAs as a result of abortive transcriptioninitiation events, double stranded RNA generated by RNA dependent RNApolymerase activity, RNA-primed transcription from RNA templates, and/orRNA comprising a self-complementary 3′ extension region, may be removedby purification, including purification by reverse phase chromatography.It may be advantageous to eliminate the need for purification by reversephase chromatography during production of a composition including RNA;therefore, there is a need for strategies to minimizing RNA impuritieswithout reverse phase purification.

In some embodiments, RNA impurities may be minimized by using5-methoxy-uridine as the uridine source for in vitro synthesis, e.g., invitro transcription with an RNA polymerase (e.g., T7 RNA polymerase). Insome embodiments, a composition including mRNA including5-methoxy-uridine has fewer RNA impurities. In some embodiments, RNAimpurities may be minimized with reverse phase by performing affinitychromatography, precipitation, membrane purification, or tangential flowfiltration (TFF) to remove the aberrant transcription products. In someembodiments, the level of aberrant transcription products in acomposition may be determined, and purification of the composition,e.g., by reverse phase chromatography performed if the level of aberranttranscription products is greater than a predetermined value.

Polypeptide Variants

Provided are nucleic acids that encode variant polypeptides, which havea certain identity with a reference polypeptide sequence. The term“identity” as known in the art, refers to a relationship between thesequences of two or more peptides, as determined by comparing thesequences. In the art, “identity” also means the degree of sequencerelatedness between peptides, as determined by the number of matchesbetween strings of two or more amino acid residues. “Identity” measuresthe percent of identical matches between the smaller of two or moresequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., “algorithms”). Identity ofrelated peptides can be readily calculated by known methods. Suchmethods include, but are not limited to, those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

In some embodiments, the polypeptide variant has the same or a similaractivity as the reference polypeptide. Alternatively, the variant has analtered activity (e.g., increased or decreased) relative to a referencepolypeptide. Generally, variants of a particular polynucleotide orpolypeptide of the present disclosure will have at least about 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more sequence identity to that particularreference polynucleotide or polypeptide as determined by sequencealignment programs and parameters described herein and known to thoseskilled in the art.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of this present disclosure. For example, providedherein is any protein fragment of a reference protein (meaning apolypeptide sequence at least one amino acid residue shorter than areference polypeptide sequence but otherwise identical) 10, 20, 30, 40,50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length Inanother example, any protein that includes a stretch of about 20, about30, about 40, about 50, or about 100 amino acids which are about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, orabout 100% identical to any of the sequences described herein can beutilized in accordance with the present disclosure. In certainembodiments, a protein sequence to be utilized in accordance with thepresent disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or moremutations as shown in any of the sequences provided or referencedherein.

Erythropoietin (EPO) and granulocyte colony-stimulating factor (GCSF)are exemplary polypeptides.

Polypeptide Libraries

Also provided are polynucleotide libraries containing nucleosidealterations, wherein the polynucleotides individually contain a firstnucleic acid sequence encoding a polypeptide, such as an antibody,protein binding partner, scaffold protein, and other polypeptides knownin the art. Preferably, the polynucleotides are mRNA in a form suitablefor direct introduction into a target cell host, which in turnsynthesizes the encoded polypeptide.

In certain embodiments, multiple variants of a protein, each withdifferent amino acid alteration(s), are produced and tested to determinethe best variant in terms of pharmacokinetics, stability,biocompatibility, and/or biological activity, or a biophysical propertysuch as expression level. Such a library may contain 10, 10², 10³, 10⁴,10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or over 10⁹ possible variants (includingsubstitutions, deletions of one or more residues, and insertion of oneor more residues).

Polypeptide-Nucleic Acid Complexes

Proper protein translation involves the physical aggregation of a numberof polypeptides and nucleic acids associated with the mRNA. Provided bythe present disclosure are protein-nucleic acid complexes, containing atranslatable mRNA having one or more nucleoside alterations (e.g., atleast two different nucleoside alterations) and one or more polypeptidesbound to the mRNA. Generally, the proteins are provided in an amounteffective to prevent or reduce an innate immune response of a cell intowhich the complex is introduced.

Synthesis of Alternative Nucleic Acids

Nucleic acids for use in accordance with the present disclosure may beprepared according to any available technique including, but not limitedto chemical synthesis, enzymatic synthesis, which is generally termed invitro transcription, enzymatic or chemical cleavage of a longerprecursor, etc. Methods of synthesizing RNAs are known in the art (see,e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach,Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn,P. (ed.) Oligonucleotide synthesis: methods and applications, Methods inMolecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press,2005; both of which are incorporated herein by reference).

In certain embodiments, a method for producing an mRNA encoding apolypeptide of interest comprises contacting a cDNA that encodes theprotein of interest with an RNA polymerase in the presence of anucleotide triphosphate mix, wherein at least 90% (e.g., at least 95% or100%) of the uracils are 5-methoxy-uracil. The invention also providesmRNA produced by such methods. The methods may include additional steps,such as capping (e.g. the addition of a 5′-cap structure), addition of apoly-A tail and/or formulation into a pharmaceutical composition. TheRNA polymerase may be T7 RNA polymerase. The in vitro transcriptionreaction mixture may include a transcription buffer (such as 400 mMTris-HCl pH 8.0, or an equivalent) and may include MgCl₂, DTT,Spermidine (or equivalents). An RNase inhibitor may be included. Theremaining reaction volume is generally made up with dH₂O. The reactionmay be incubated at approximately 37° C. (such as between 30 and 40° C.)and may be incubated for 3 hr-5 hrs (such as 3½ hr-4½ hr, or about 4hr). The RNA may then be cleaned using DNase and a purification kit.

Alternative nucleic acids need not be uniformly present along the entirelength of the molecule. Different nucleotide alterations and/or backbonestructures may exist at various positions in the nucleic acid. One ofordinary skill in the art will appreciate that the nucleotide analogs orother alteration(s) may be located at any position(s) of a nucleic acidsuch that the function of the nucleic acid is not substantiallydecreased. An alteration may also be a 5′ or 3′ terminal alteration. Thenucleic acids may contain at a minimum one and at maximum 100%alternative nucleotides, or any intervening percentage, such as at least5% alternative nucleotides, at least 10% alternative nucleotides, atleast 25% alternative nucleotides, at least 50% alternative nucleotides,at least 80% alternative nucleotides, or at least 90% alternativenucleotides. For example, the nucleic acids may contain an alternativepyrimidine such as uracil or cytosine. In some embodiments, at least 5%,at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or100% of the uracil in the nucleic acid is replaced with an alternativeuracil. The alternative uracil can be replaced by a compound having asingle unique structure, or can be replaced by a plurality of compoundshaving different structures (e.g., 2, 3, 4 or more unique structures).In some embodiments, at least 5%, at least 10%, at least 25%, at least50%, at least 80%, at least 90% or 100% of the cytosine in the nucleicacid is replaced with an alternative cytosine. The alternative cytosinecan be replaced by a compound having a single unique structure, or canbe replaced by a plurality of compounds having different structures(e.g., 2, 3, 4 or more unique structures).

Generally, the shortest length of an alternative mRNA of the presentdisclosure can be the length of an mRNA sequence that is sufficient toencode for a dipeptide. In another embodiment, the length of the mRNAsequence is sufficient to encode for a tripeptide. In anotherembodiment, the length of an mRNA sequence is sufficient to encode for atetrapeptide. In another embodiment, the length of an mRNA sequence issufficient to encode for a pentapeptide. In another embodiment, thelength of an mRNA sequence is sufficient to encode for a hexapeptide. Inanother embodiment, the length of an mRNA sequence is sufficient toencode for a heptapeptide. In another embodiment, the length of an mRNAsequence is sufficient to encode for an octapeptide. In anotherembodiment, the length of an mRNA sequence is sufficient to encode for anonapeptide. In another embodiment, the length of an mRNA sequence issufficient to encode for a decapeptide.

Examples of dipeptides that the alternative nucleic acid sequences canencode for include, but are not limited to, carnosine and anserine.

In a further embodiment, the mRNA is greater than 30 nucleotides inlength. In another embodiment, the RNA molecule is greater than 35nucleotides in length. In another embodiment, the length is at least 40nucleotides. In another embodiment, the length is at least 45nucleotides. In another embodiment, the length is at least 55nucleotides. In another embodiment, the length is at least 50nucleotides. In another embodiment, the length is at least 60nucleotides. In another embodiment, the length is at least 80nucleotides. In another embodiment, the length is at least 90nucleotides. In another embodiment, the length is at least 100nucleotides. In another embodiment, the length is at least 120nucleotides. In another embodiment, the length is at least 140nucleotides. In another embodiment, the length is at least 160nucleotides. In another embodiment, the length is at least 180nucleotides. In another embodiment, the length is at least 200nucleotides. In another embodiment, the length is at least 250nucleotides. In another embodiment, the length is at least 300nucleotides. In another embodiment, the length is at least 350nucleotides. In another embodiment, the length is at least 400nucleotides. In another embodiment, the length is at least 450nucleotides. In another embodiment, the length is at least 500nucleotides. In another embodiment, the length is at least 600nucleotides. In another embodiment, the length is at least 700nucleotides. In another embodiment, the length is at least 800nucleotides. In another embodiment, the length is at least 900nucleotides. In another embodiment, the length is at least 1000nucleotides. In another embodiment, the length is at least 1100nucleotides. In another embodiment, the length is at least 1200nucleotides. In another embodiment, the length is at least 1300nucleotides. In another embodiment, the length is at least 1400nucleotides. In another embodiment, the length is at least 1500nucleotides. In another embodiment, the length is at least 1600nucleotides. In another embodiment, the length is at least 1800nucleotides. In another embodiment, the length is at least 2000nucleotides. In another embodiment, the length is at least 2500nucleotides. In another embodiment, the length is at least 3000nucleotides. In another embodiment, the length is at least 4000nucleotides. In another embodiment, the length is at least 5000nucleotides, or greater than 5000 nucleotides.

For example, the alternative nucleic acids described herein can beprepared using methods that are known to those skilled in the art ofnucleic acid synthesis.

In some embodiments, the present disclosure provides for methods ofsynthesizing a pharmaceutical nucleic acid, comprising the steps of:

a) providing a complementary deoxyribonucleic acid (cDNA) that encodes apharmaceutical protein of interest;

b) selecting a nucleotide and

c) contacting the provided cDNA and the selected nucleotide with an RNApolymerase, under conditions such that the pharmaceutical nucleic acidis synthesized.

In further embodiments, the pharmaceutical nucleic acid is a ribonucleicacid (RNA).

In still a further aspect of the present disclosure, the alternativenucleic acids can be prepared using solid phase synthesis methods.

5′-Capping

The 5′-cap structure of an mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns removal during mRNA splicing.

Endogenous mRNA molecules may be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA. This5′-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA mayoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure may target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

Alterations to the nucleic acids of the present invention may generate anon-hydrolyzable cap structure preventing decapping and thus increasingmRNA half-life. Because cap structure hydrolysis requires cleavage of5′-ppp-5′ phosphorodiester linkages, alternative nucleotides may be usedduring the capping reaction. For example, a Vaccinia Capping Enzyme fromNew England Biolabs (Ipswich, Mass.) may be used with α-thio-guanosinenucleotides according to the manufacturer's instructions to create aphosphorothioate linkage in the 5′-ppp-5′ cap. Additional alternativeguanosine nucleotides may be used such as α-methyl-phosphonate andseleno-phosphate nucleotides.

Additional alterations include, but are not limited to, 2′-O-methylationof the ribose sugars of 5′-terminal and/or 5′-anteterminal nucleotidesof the mRNA (as mentioned above) on the 2′-hydroxyl group of the sugarring. Multiple distinct 5′-cap structures can be used to generate the5′-cap of a nucleic acid molecule, such as an mRNA molecule.

5′-cap structures include those described in International PatentPublication Nos. WO2008127688, WO 2008016473, and WO 2011015347, each ofwhich is incorporated herein by reference in its entirety.

Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e. endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs may be chemically (i.e. non-enzymatically) orenzymatically synthesized and/linked to a nucleic acid molecule.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanosines linked by a 5′-5′-triphosphate group, wherein one guanosinecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which may equivalently be designated 3′-O-Me-m7G(5′)ppp(5′)G). The 3′-Oatom of the other, unaltered, guanosine becomes linked to the5′-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNAor mmRNA). The N7- and 3′-O-methlyated guanosine provides the terminalmoiety of the capped nucleic acid molecule (e.g. mRNA or mmRNA).

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, mtm-ppp-G).

In one embodiment, the cap is a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog may be modified atdifferent phosphate positions with a boranophosphate group or aphophoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the cap analog is a N7-(4-chlorophenoxyethyl)substituted dicnucleotide form of a cap analog known in the art and/ordescribed herein. Non-limiting examples of a N7-(4-chlorophenoxyethyl)substituted dinucleotide form of a cap analog include aN7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m3′-OG(5′)ppp(5′)G cap analog (See e.g., thevarious cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by referencein its entirety). In another embodiment, a cap analog of the presentinvention is a 4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a nucleic acidmolecule in an in vitro transcription reaction, up to 20% of transcriptsremain uncapped. This, as well as the structural differences of a capanalog from endogenous 5′-cap structures of nucleic acids produced bythe endogenous, cellular transcription machinery, may lead to reducedtranslational competency and reduced cellular stability.

Alternative nucleic acids of the invention may also be cappedpost-transcriptionally, using enzymes, in order to generate moreauthentic 5′-cap structures. As used herein, the phrase “more authentic”refers to a feature that closely mirrors or mimics, either structurallyor functionally, an endogenous or wild-type feature. That is, a “moreauthentic” feature is better representative of an endogenous, wild-type,natural or physiological cellular function and/or structure as comparedto synthetic features or analogs, etc., of the prior art, or whichoutperforms the corresponding endogenous, wild-type, natural orphysiological feature in one or more respects. Non-limiting examples ofmore authentic 5′-cap structures of the present invention are thosewhich, among other things, have enhanced binding of cap bindingproteins, increased half life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping, as compared to synthetic5′-cap structures known in the art (or to a wild-type, natural orphysiological 5′-cap structure). For example, recombinant Vaccinia VirusCapping Enzyme and recombinant 2′-O-methyltransferase enzyme can createa canonical 5′-5′-triphosphate linkage between the 5′-terminalnucleotide of an mRNA and a guanosine cap nucleotide wherein the capguanosine contains an N7 methylation and the 5′-terminal nucleotide ofthe mRNA contains a 2′-O-methyl. Such a structure is termed the Cap1structure. This cap results in a higher translational-competency andcellular stability and a reduced activation of cellular pro-inflammatorycytokines, as compared, e.g., to other 5′cap analog structures known inthe art. Cap structures include 7mG(5′)ppp(5′)N,pN2p (cap 0),7mG(5′)ppp(5′)NImpNp (cap 1), 7mG(5′)-ppp(5′)NImpN2mp (cap 2) andm(7)Gpppm(3)(6,6,2′)Apm(2′)Apm(2′)Cpm(2)(3,2′)Up (cap 4).

Because the alternative nucleic acids may be cappedpost-transcriptionally, and because this process is more efficient,nearly 100% of the alternative nucleic acids may be capped. This is incontrast to −80% when a cap analog is linked to an mRNA in the course ofan in vitro transcription reaction.

According to the present invention, 5′ terminal caps may includeendogenous caps or cap analogs. According to the present invention, a 5′terminal cap may comprise a guanosine analog. Useful guanosine analogsinclude inosine, N1-methyl-guanosine, 2′fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,and 2-azido-guanosine.

In one embodiment, the nucleic acids described herein may contain amodified 5′-cap. A modification on the 5′-cap may increase the stabilityof mRNA, increase the half-life of the mRNA, and could increase the mRNAtranslational efficiency. The modified 5′-cap may include, but is notlimited to, one or more of the following modifications: modification atthe 2′ and/or 3′ position of a capped guanosine triphosphate (GTP), areplacement of the sugar ring oxygen (that produced the carbocyclicring) with a methylene moiety (CH₂), a modification at the triphosphatebridge moiety of the cap structure, or a modification at the nucleobase(G) moiety.

The 5′-cap structure that may be modified includes, but is not limitedto, the caps described herein such as Cap0 having the substratestructure for cap dependent translation of:

or Cap1 having the substrate structure for cap dependent translation of:

As a non-limiting example, the modified 5′-cap may have the substratestructure for cap dependent translation of:

where R₁ and R₂ are defined in Table 5:

TABLE 5 R₁ and R₂ groups for CAP-022 to CAP096. Cap Struc- ture NumberR₁ R₂ CAP-022 C₂H₅ (Ethyl) H CAP-023 H C₂H₅ (Ethyl) CAP-024 C₂H₅ (Ethyl)C₂H₅ (Ethyl) CAP-025 C₃H₇ (Propyl) H CAP-026 H C₃H₇ (Propyl) CAP-027C₃H₇ (Propyl) C₃H₇ (Propyl) CAP-028 C₄H₉ (Butyl) H CAP-029 H C₄H₉(Butyl) CAP-030 C₄H₉ (Butyl) C₄H₉ (Butyl) CAP-031 C₅H₁₁ (Pentyl) HCAP-032 H C₅H₁₁ (Pentyl) CAP-033 C₅H₁₁ (Pentyl) C₅H₁₁ (Pentyl) CAP-034H₂C—C≡CH H (Propargyl) CAP-035 H H₂C—C≡CH (Propargyl) CAP-036 H₂C—C≡CHH₂C—C≡CH (Propargyl) (Propargyl) CAP-037 CH₂CH═CH₂ (Allyl) H CAP-038 HCH₂CH═CH₂ (Allyl) CAP-039 CH₂CH═CH₂ (Allyl) CH₂CH═CH₂ (Allyl) CAP-040CH₂OCH₃ (MOM) H CAP-041 H CH₂OCH₃ (MOM) CAP-042 CH₂OCH₃ (MOM) CH₂OCH₃(MOM) CAP-043 CH₂OCH₂CH₂OCH₃ (MEM) H CAP-044 H CH₂OCH₂CH₂OCH₃ (MEM)CAP-045 CH₂OCH₂CH₂OCH₃ (MEM) CH₂OCH₂CH₂OCH₃ (MEM) CAP-046 CH₂SCH₃ (MTM)H CAP-047 H CH₂SCH₃ (MTM) CAP-048 CH₂SCH₃ (MTM) CH₂SCH₃ (MTM) CAP-049CH₂C₆H₅ (Benzyl) H CAP-050 H CH₂C₆H₅ (Benzyl) CAP-051 CH₂C₆H₅ (Benzyl)CH₂C₆H₅ (Benzyl) CAP-052 CH₂OCH₂C₆H₅ (BOM) H CAP-053 H CH₂OCH₂C₆H₅ (BOM)CAP-054 CH₂OCH₂C₆H₅ (BOM) CH₂OCH₂C₆H₅ (BOM) CAP-055 CH₂C₆H₄—OMe H(p-Methoxybenzyl) CAP-056 H CH₂C₆H₄—OMe (p-Methoxybenzyl) CAP-057CH₂C₆H₄—OMe CH₂C₆H₄—OMe (p-Methoxybenzyl) (p-Methoxybenzyl) CAP-058CH₂C₆H₄—NO₂ H (p-Nitrobenzyl) CAP-059 H CH₂C₆H₄—NO₂ (p-Nitrobenzyl)CAP-060 CH₂C₆H₄—NO₂ CH₂C₆H₄—NO₂ (p-Nitrobenzyl) (p-Nitrobenzyl) CAP-061CH₂C₆H₄—X (p-Halobenzyl) H where X = F, Cl, Br or I CAP-062 H CH₂C₆H₄—X(p-Halobenzyl) where X = F, Cl, Br or I CAP-063 CH₂C₆H₄—X (p-Halobenzyl)CH₂C₆H₄—X (p-Halobenzyl) where X = F, Cl, Br or I where X = F, Cl, Br orI CAP-064 CH₂C₆H₄—N₃ (p- H Azidobenzyl) CAP-065 H CH₂C₆H₄—N₃ (p-Azidobenzyl) CAP-066 CH₂C₆H₄—N₃ (p- CH₂C₆H₄—N₃ (p- Azidobenzyl)Azidobenzyl) CAP-067 CH₂C₆H₄—CF₃ (p- H Trifluoromethylbenzyl) CAP-068 HCH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl) CAP-069 CH₂C₆H₄—CF₃ (p-CH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl) Trifluoromethylbenzyl) CAP-070CH₂C₆H₄—OCF₃ (p- H Trifluoromethoxylbenzyl) CAP-071 H CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) CAP-072 CH₂C₆H₄—OCF₃ (p- CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) Trifluoromethoxylbenzyl) CAP-073 CH₂C₆H₃—(CF₃)₂[2,4- H bis(Trifluoromethyl)benzyl] CAP-074 H CH₂C₆H₃—(CF₃)₂ [2,4-bis(Trifluoromethyl)benzyl] CAP-075 CH₂C₆H₃—(CF₃)₂ [2,4- CH₂C₆H₃—(CF₃)₂[2,4- bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-076Si(C₆H₅)₂C₄H₉ (t- H Butyldiphenylsilyl) CAP-077 H Si(C₆H₅)₂C₄H₉ (t-Butyldiphenylsilyl) CAP-078 Si(C₆H₅)₂C₄H₉ (t- Si(C₆H₅)₂C₄H₉ (t-Butyldiphenylsilyl) Butyldiphenylsilyl) CAP-079 CH₂CH₂CH═CH₂ H(Homoallyl) CAP-080 H CH₂CH₂CH═CH₂ (Homoallyl) CAP-081 CH₂CH₂CH═CH₂CH₂CH₂CH═CH₂ (Homoallyl) (Homoallyl) CAP-082 P(O)(OH)₂ (MP) H CAP-083 HP(O)(OH)₂ (MP) CAP-084 P(O)(OH)₂ (MP) P(O)(OH)₂ (MP) CAP-085 P(S)(OH)₂(Thio-MP) H CAP-086 H P(S)(OH)₂ (Thio-MP) CAP-087 P(S)(OH)₂ (Thio-MP)P(S)(OH)₂ (Thio-MP) CAP-088 P(O)(CH₃)(OH) H (Methylphophonate) CAP-089 HP(O)(CH₃)(OH) (Methylphophonate) CAP-090 P(O)(CH₃)(OH) P(O)(CH₃)(OH)(Methylphophonate) (Methylphophonate) CAP-091 PN(^(I)Pr)₂(OCH₂CH₂CN) H(Phosporamidite) CAP-092 H PN(^(I)Pr)₂(OCH₂CH₂CN) (Phosporamidite)CAP-093 PN(^(I)Pr)₂(OCH₂CH₂CN) PN(^(I)Pr)₂(OCH₂CH₂CN) (Phosporamidite)(Phosporamidite) CAP-094 SO₂CH₃ H (Methanesulfonic acid) CAP-095 HSO₂CH₃ (Methanesulfonic acid) CAP-096 SO₂CH₃ SO₂CH₃ (Methanesulfonicacid) (Methanesulfonic acid)

or where R₁ and R₂ are defined in Table 6:

TABLE 6 R₁ and R₂ groups for CAP-097 to CAP111. Cap Structure Number R₁R₂ CAP-097 NH₂ (amino) H CAP-098 H NH₂ (amino) CAP-099 NH₂ (amino) NH₂(amino) CAP-100 N₃ (Azido) H CAP-101 H N₃ (Azido) CAP-102 N₃ (Azido) N₃(Azido) CAP-103 X (Halo: F, Cl, Br, I) H CAP-104 H X (Halo: F, Cl, Br,I) CAP-105 X (Halo: F, Cl, Br, I) X (Halo: F, Cl, Br, I) CAP-106 SH(Thiol) H CAP-107 H SH (Thiol) CAP-108 SH (Thiol) SH (Thiol) CAP-109SCH₃ (Thiomethyl) H CAP-110 H SCH₃ (Thiomethyl) CAP-111 SCH₃(Thiomethyl) SCH₃ (Thiomethyl)

In Table 5, “MOM” stands for methoxymethyl, “MEM” stands formethoxyethoxymethyl, “MTM” stands for methylthiomethyl, “BOM” stands forbenzyloxymethyl and “MP” stands for monophosphonate.

In a non-limiting example, the modified 5′-cap may have the substratestructure for vaccinia mRNA capping enzyme of:

where R₁ and R₂ are defined in Table 7:

TABLE 7 R₁ and R₂ groups for CAP-136 to CAP-210. Cap Struc- ture NumberR₁ R₂ CAP-136 C₂H₅ (Ethyl) H CAP-137 H C₂H₅ (Ethyl) CAP-138 C₂H₅ (Ethyl)C₂H₅ (Ethyl) CAP-139 C₃H₇ (Propyl) H CAP-140 H C₃H₇ (Propyl) CAP-141C₃H₇ (Propyl) C₃H₇ (Propyl) CAP-142 C₄H₉ (Butyl) H CAP-143 H C₄H₉(Butyl) CAP-144 C₄H₉ (Butyl) C₄H₉ (Butyl) CAP-145 C₅H₁₁ (Pentyl) HCAP-146 H C₅H₁₁ (Pentyl) CAP-147 C₅H₁₁ (Pentyl) C₅H₁₁ (Pentyl) CAP-148H₂C—C≡CH H (Propargyl) CAP-149 H H₂C—C≡CH (Propargyl) CAP-150 H₂C—C≡CHH₂C—C≡CH (Propargyl) (Propargyl) CAP-151 CH₂CH═CH₂ (Allyl) H CAP-152 HCH₂CH═CH₂ (Allyl) CAP-153 CH₂CH═CH₂ (Allyl) CH₂CH═CH₂ (Allyl) CAP-154CH₂OCH₃ (MOM) H CAP-155 H CH₂OCH₃ (MOM) CAP-156 CH₂OCH₃ (MOM) CH₂OCH₃(MOM) CAP-157 CH₂OCH₂CH₂OCH₃ (MEM) H CAP-158 H CH₂OCH₂CH₂OCH₃ (MEM)CAP-159 CH₂OCH₂CH₂OCH₃ (MEM) CH₂OCH₂CH₂OCH₃ (MEM) CAP-160 CH₂SCH₃ (MTM)H CAP-161 H CH₂SCH₃ (MTM) CAP-162 CH₂SCH₃ (MTM) CH₂SCH₃ (MTM) CAP-163CH₂C₆H₅ (Benzyl) H CAP-164 H CH₂C₆H₅ (Benzyl) CAP-165 CH₂C₆H₅ (Benzyl)CH₂C₆H₅ (Benzyl) CAP-166 CH₂OCH₂C₆H₅ (BOM) H CAP-167 H CH₂OCH₂C₆H₅ (BOM)CAP-168 CH₂OCH₂C₆H₅ (BOM) CH₂OCH₂C₆H₅ (BOM) CAP-169 CH₂C₆H₄—OMe (p- HMethoxybenzyl) CAP-170 H CH₂C₆H₄—OMe (p- Methoxybenzyl) CAP-171CH₂C₆H₄—OMe (p- CH₂C₆H₄—OMe (p- Methoxybenzyl) Methoxybenzyl) CAP-172CH₂C₆H₄—NO₂ (p- H Nitrobenzyl) CAP-173 H CH₂C₆H₄—NO₂ (p- Nitrobenzyl)CAP-174 CH₂C₆H₄—NO₂ (p- CH₂C₆H₄—NO₂ (p- Nitrobenzyl) Nitrobenzyl)CAP-175 CH₂C₆H₄—X (p-Halobenzyl) H where X = F, Cl, Br or I CAP-176 HCH₂C₆H₄—X (p-Halobenzyl) where X = F, Cl, Br or I CAP-177 CH₂C₆H₄—X(p-Halobenzyl) CH₂C₆H₄—X (p-Halobenzyl) where X = F, Cl, Br or I where X= F, Cl, Br or I CAP-178 CH₂C₆H₄—N₃ (p- H Azidobenzyl) CAP-179 HCH₂C₆H₄—N₃ (p- Azidobenzyl) CAP-180 CH₂C₆H₄—N₃ (p- CH₂C₆H₄—N₃ (p-Azidobenzyl) Azidobenzyl) CAP-181 CH₂C₆H₄—CF₃ (p- HTrifluoromethylbenzyl) CAP-182 H CH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl)CAP-183 CH₂C₆H₄—CF₃ (p- CH₂C₆H₄—CF₃ (p- Trifluoromethylbenzyl)Trifluoromethylbenzyl) CAP-184 CH₂C₆H₄—OCF₃ (p- HTrifluoromethoxylbenzyl) CAP-185 H CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) CAP-186 CH₂C₆H₄—OCF₃ (p- CH₂C₆H₄—OCF₃ (p-Trifluoromethoxylbenzyl) Trifluoromethoxylbenzyl) CAP-187 CH₂C₆H₃—(CF₃)₂[2,4- H bis(Trifluoromethyl)benzyl] CAP-188 H CH₂C₆H₃—(CF₃)₂ [2,4-bis(Trifluoromethyl)benzyl] CAP-189 CH₂C₆H₃—(CF₃)₂ [2,4- CH₂C₆H₃—(CF₃)₂[2,4- bis(Trifluoromethyl)benzyl] bis(Trifluoromethyl)benzyl] CAP-190Si(C₆H₅)₂C₄H₉ (t- H Butyldiphenylsilyl) CAP-191 H Si(C₆H₅)₂C₄H₉ (t-Butyldiphenylsilyl) CAP-192 Si(C₆H₅)₂C₄H₉ (t- Si(C₆H₅)₂C₄H₉ (t-Butyldiphenylsilyl) Butyldiphenylsilyl) CAP-193 CH₂CH₂CH═CH₂ H(Homoallyl) CAP-194 H CH₂CH₂CH═CH₂ (Homoallyl) CAP-195 CH₂CH₂CH═CH₂CH₂CH₂CH═CH₂ (Homoallyl) (Homoallyl) CAP-196 P(O)(OH)₂ (MP) H CAP-197 HP(O)(OH)₂ (MP) CAP-198 P(O)(OH)₂ (MP) P(O)(OH)₂ (MP) CAP-199 P(S)(OH)₂(Thio-MP) H CAP-200 H P(S)(OH)₂ (Thio-MP) CAP-201 P(S)(OH)₂ (Thio-MP)P(S)(OH)₂ (Thio-MP) CAP-202 P(O)(CH₃)(OH) H (Methylphophonate) CAP-203 HP(O)(CH₃)(OH) (Methylphophonate) CAP-204 P(O)(CH₃)(OH) P(O)(CH₃)(OH)(Methylphophonate) (Methylphophonate) CAP-205 PN(^(I)Pr)₂(OCH₂CH₂CN) H(Phosporamidite) CAP-206 H PN(^(I)Pr)₂(OCH₂CH₂CN) (Phosporamidite)CAP-207 PN(^(I)Pr)₂(OCH₂CH₂CN) PN(^(I)Pr)₂(OCH₂CH₂CN) (Phosporamidite)(Phosporamidite) CAP-208 SO₂CH₃ H (Methanesulfonic acid) CAP-209 HSO₂CH₃ (Methanesulfonic acid) CAP-210 SO₂CH₃ SO₂CH₃ (Methanesulfonicacid) (Methanesulfonic acid)

or where R₁ and R₂ are defined in Table 8:

TABLE 8 R₁ and R₂ groups for CAP-211 to 225. Cap Structure Number R₁ R₂CAP-211 NH₂ (amino) H CAP-212 H NH₂ (amino) CAP-213 NH₂ (amino) NH₂(amino) CAP-214 N₃ (Azido) H CAP-215 H N₃ (Azido) CAP-216 N₃ (Azido) N₃(Azido) CAP-217 X (Halo: F, Cl, Br, I) H CAP-218 H X (Halo: F, Cl, Br,I) CAP-219 X (Halo: F, Cl, Br, I) X (Halo: F, Cl, Br, I) CAP-220 SH(Thiol) H CAP-221 H SH (Thiol) CAP-222 SH (Thiol) SH (Thiol) CAP-223SCH₃ (Thiomethyl) H CAP-224 H SCH₃ (Thiomethyl) CAP-225 SCH₃(Thiomethyl) SCH₃ (Thiomethyl)

In Table 7, “MOM” stands for methoxymethyl, “MEM” stands formethoxyethoxymethyl, “MTM” stands for methylthiomethyl, “BOM” stands forbenzyloxymethyl and “MP” stands for monophosphonate.

In another non-limiting example, of the modified capping structuresubstrates CAP-112-CAP-225 could be added in the presence of vacciniacapping enzyme with a component to create enzymatic activity such as,but not limited to, S-adenosylmethionine (AdoMet), to form a modifiedcap for mRNA.

In one embodiment, the replacement of the sugar ring oxygen (thatproduced the carbocyclic ring) with a methylene moiety (CH₂) couldcreate greater stability to the C—N bond against phosphorylases as theC—N bond is resitant to acid or enzymatic hydrolysis. The methylenemoiety may also increase the stability of the triphosphate bridge moietyand thus increasing the stability of the mRNA. As a non-limitingexample, the cap substrate structure for cap dependent translation mayhave the structure such as, but not limited to, CAP-014 and CAP-015and/or the cap substrate structure for vaccinia mRNA capping enzyme suchas, but not limited to, CAP-123 and CAP-124. In another example,CAP-112-CAP-122 and/or CAP-125-CAP-225, can be modified by replacing thesugar ring oxygen (that produced the carbocyclic ring) with a methylenemoiety (CH₂).

In another embodiment, the triphophosphate bridge may be modified by thereplacement of at least one oxygen with sulfur (thio), a borane (BH₃)moiety, a methyl group, an ethyl group, a methoxy group and/orcombinations thereof. This modification could increase the stability ofthe mRNA towards decapping enzymes. As a non-limiting example, the capsubstrate structure for cap dependent translation may have the structuresuch as, but not limited to, CAP-016-CAP-021 and/or the cap substratestructure for vaccinia mRNA capping enzyme such as, but not limited to,CAP-125-CAP-130. In another example, CAP-003-CAP-015, CAP-022-CAP-124and/or CAP-131-CAP-225, can be modified on the triphosphate bridge byreplacing at least one of the triphosphate bridge oxygens with sulfur(thio), a borane (BH₃) moiety, a methyl group, an ethyl group, a methoxygroup and/or combinations thereof.

In one embodiment, CAP-001-134 and/or CAP-136-CAP-225 may be modified tobe a thioguanosine analog similar to CAP-135. The thioguanosine analogmay comprise additional modifications such as, but not limited to, amodification at the triphosphate moiety (e.g., thio, BH₃, CH₃, C₂H₅,OCH₃, S and S with OCH₃), a modification at the 2′ and/or 3′ positionsof 6-thio guanosine as described herein and/or a replacement of thesugar ring oxygen (that produced the carbocyclic ring) as describedherein.

In one embodiment, CAP-001-121 and/or CAP-123-CAP-225 may be modified tobe a modified 5′-cap similar to CAP-122. The modified 5′-cap maycomprise additional modifications such as, but not limited to, amodification at the triphosphate moiety (e.g., thio, BH₃, CH₃, C₂H₅,OCH₃, S and S with OCH₃), a modification at the 2′ and/or 3′ positionsof 6-thio guanosine as described herein and/or a replacement of thesugar ring oxygen (that produced the carbocyclic ring) as describedherein.

In one embodiment, the 5′-cap modification may be the attachment ofbiotin or conjugation at the 2′ or 3′ position of a GTP.

In another embodiment, the 5′-cap modification may include a CF₂modified triphosphate moiety.

In another embodiment, the triphosphate bridge of any of the capstructures described herein may be replaced with a tetraphosphate orpentaphosphate bridge. Examples of tetraphosphate and pentaphosphatecontaining bridges and other cap modifications are described inJemielity, J. et al. RNA 2003 9:1108-1122; Grudzien-Nogalska, E. et al.Methods Mol. Biol. 2013 969:55-72; and Grudzien, E. et al. RNA, 200410:1479-1487, each of which is incorporated herein by reference in itsentirety.

Terminal Architecture Alterations: Stem Loop

In one embodiment, the nucleic acids of the present invention mayinclude a stem loop such as, but not limited to, a histone stem loop.The stem loop may be a nucleotide sequence that is about 25 or about 26nucleotides in length such as, but not limited to, SEQ ID NOs: 7-17 asdescribed in International Patent Publication No. WO2013103659,incorporated herein by reference in its entirety. The histone stem loopmay be located 3′ relative to the coding region (e.g., at the 3′terminus of the coding region). As a non-limiting example, the stem loopmay be located at the 3′ end of a nucleic acid described herein.

In one embodiment, the stem loop may be located in the second terminalregion. As a non-limiting example, the stem loop may be located withinan untranslated region (e.g., 3′3′-UTR) in the second terminal region.

In one embodiment, the nucleic acid such as, but not limited to mRNA,which comprises the histone stem loop may be stabilized by the additionof at least one chain terminating nucleoside. Not wishing to be bound bytheory, the addition of at least one chain terminating nucleoside mayslow the degradation of a nucleic acid and thus can increase thehalf-life of the nucleic acid.

In one embodiment, the chain terminating nucleoside may be, but is notlimited to, those described in International Patent Publication No.WO2013103659, incorporated herein by reference in its entirety. Inanother embodiment, the chain terminating nucleosides which may be usedwith the present invention includes, but is not limited to,3′-deoxyadenosine (cordycepin), 3′-deoxyuridine, 3′-deoxycytidine,3′-deoxyguanosine, 3′-deoxythymidine, 2′,3′-dideoxynucleosides, such as2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytidine,2′,3′-dideoxyguanosine, 2′,3′-dideoxythymidine, a 2′-deoxynucleoside, ora 2′ or 3′-O-methylnucleoside.

In another embodiment, the nucleic acid such as, but not limited tomRNA, which comprises the histone stem loop may be stabilized by analteration to the 3′-region of the nucleic acid that can prevent and/orinhibit the addition of oligio(U) (see e.g., International PatentPublication No. WO2013103659, incorporated herein by reference in itsentirety).

In yet another embodiment, the nucleic acid such as, but not limited tomRNA, which comprises the histone stem loop may be stabilized by theaddition of an oligonucleotide that terminates in a 3′-deoxynucleoside,2′,3′-dideoxynucleoside 3′-O-methyl nucleosides, 3′-O-ethyl nucleosides,3′-arabinosides, and other alternative nucleosides known in the artand/or described herein.

In one embodiment, the nucleic acids of the present invention mayinclude a histone stem loop, a polyA tail sequence and/or a 5′-capstructure. The histone stem loop may be before and/or after the polyAtail sequence. The nucleic acids comprising the histone stem loop and apolyA tail sequence may include a chain terminating nucleoside describedherein.

In another embodiment, the nucleic acids of the present invention mayinclude a histone stem loop and a 5′-cap structure. The 5′-cap structuremay include, but is not limited to, those described herein and/or knownin the art.

In one embodiment, the conserved stem loop region may comprise a miRsequence described herein. As a non-limiting example, the stem loopregion may comprise the seed sequence of a miR sequence describedherein. In another non-limiting example, the stem loop region maycomprise a miR-122 seed sequence.

In another embodiment, the conserved stem loop region may comprise a miRsequence described herein and may also include a TEE sequence.

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′-UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, herein incorporated byreference in its entirety).

In one embodiment, the alternative nucleic acids described herein maycomprise at least one histone stem-loop and a polyA sequence orpolyadenylation signal. Non-limiting examples of nucleic acid sequencesencoding for at least one histone stem-loop and a polyA sequence or apolyadenylation signal are described in International Patent PublicationNo. WO2013120497, WO2013120629, WO2013120500, WO2013120627,WO2013120498, WO2013120626, WO2013120499 and WO2013120628, the contentsof each of which are incorporated herein by reference in their entirety.In one embodiment, the nucleic acid encoding for a histone stem loop anda polyA sequence or a polyadenylation signal may code for a pathogenantigen or fragment thereof such as the nucleic acid sequences describedin International Patent Publication No WO2013120499 and WO2013120628,the contents of both of which are incorporated herein by reference intheir entirety. In another embodiment, the nucleic acid encoding for ahistone stem loop and a polyA sequence or a polyadenylation signal maycode for a therapeutic protein such as the nucleic acid sequencesdescribed in International Patent Publication No WO2013120497 andWO2013120629, the contents of both of which are incorporated herein byreference in their entirety. In one embodiment, the nucleic acidencoding for a histone stem loop and a polyA sequence or apolyadenylation signal may code for a tumor antigen or fragment thereofsuch as the nucleic acid sequences described in International PatentPublication No WO2013120500 and WO2013120627, the contents of both ofwhich are incorporated herein by reference in their entirety. In anotherembodiment, the nucleic acid encoding for a histone stem loop and apolyA sequence or a polyadenylation signal may code for an allergenicantigen or an autoimmune self-antigen such as the nucleic acid sequencesdescribed in International Patent Publication No WO2013120498 andWO2013120626, the contents of both of which are incorporated herein byreference in their entirety.

Terminal Architecture Alterations: 3′-UTR and Triple Helices

In one embodiment, nucleic acids of the present invention may include atriple helix on the 3′ end of the alternative nucleic acid, enhancedalternative RNA or ribonucleic acid. The 3′ end of the nucleic acids ofthe present invention may include a triple helix alone or in combinationwith a Poly-A tail.

In one embodiment, the nucleic acid of the present invention maycomprise at least a first and a second U-rich region, a conserved stemloop region between the first and second region and an A-rich region.The first and second U-rich region and the A-rich region may associateto form a triple helix on the 3′ end of the nucleic acid. This triplehelix may stabilize the nucleic acid, enhance the translationalefficiency of the nucleic acid and/or protect the 3′ end fromdegradation. Exemplary triple helices include, but are not limited to,the triple helix sequence of metastasis-associated lung adenocarcinomatranscript 1 (MALAT1), MEN-β and polyadenylated nuclear (PAN) RNA (SeeWilusz et al., Genes & Development 2012 26:2392-2407; hereinincorporated by reference in its entirety). In one embodiment, the 3′end of the alternative nucleic acids, enhanced alternative RNA orribonucleic acids of the present invention comprises a first U-richregion comprising TTTTTCTTTT (SEQ ID NO: 1), a second U-rich regioncomprising TTTTGCTTTTT (SEQ ID NO: 2) or TTTTGCTTTT (SEQ ID NO: 3), anA-rich region comprising AAAAAGCAAAA (SEQ ID NO: 4). In anotherembodiment, the 3′ end of the nucleic acids of the present inventioncomprises a triple helix formation structure comprising a first U-richregion, a conserved region, a second U-rich region and an A-rich region.

In one embodiment, the triple helix may be formed from the cleavage of aMALAT1 sequence prior to the cloverleaf structure. While not meaning tobe bound by theory, MALAT1 is a long non-coding RNA which, when cleaved,forms a triple helix and a tRNA-like cloverleaf structure. The MALAT1transcript then localizes to nuclear speckles and the tRNA-likecloverleaf localizes to the cytoplasm (Wilusz et al. Cell 2008 135(5):919-932; incorporated herein by reference in its entirety).

As a non-limiting example, the terminal end of the nucleic acid of thepresent invention comprising the MALAT1 sequence can then form a triplehelix structure, after RNaseP cleavage from the cloverleaf structure,which stabilizes the nucleic acid (Peart et al. Non-mRNA 3′ endformation: how the other half lives; WIREs RNA 2013; incorporated hereinby reference in its entirety).

In one embodiment, the nucleic acids or mRNA described herein comprise aMALAT1 sequence. In another embodiment, the nucleic acids or mRNA may bepolyadenylated. In yet another embodiment, the nucleic acids or mRNA isnot polyadenylated but has an increased resistance to degradationcompared to unaltered nucleic acids or mRNA.

In one embodiment, the nucleic acids of the present invention maycomprise a MALAT1 sequence in the second flanking region (e.g., the3′-UTR). As a non-limiting example, the MALAT1 sequence may be human ormouse.

In another embodiment, the cloverleaf structure of the MALAT1 sequencemay also undergo processing by RNaseZ and CCA adding enzyme to form atRNA-like structure called mascRNA (MALAT1-associated small cytoplasmicRNA). As a non-limiting example, the mascRNA may encode a protein or afragment thereof and/or may comprise a microRNA sequence. The mascRNAmay comprise at least one chemical alteration described herein.

Terminal Architecture Alterations: Poly-A Tails

During RNA processing, a long chain of adenosine nucleotides (poly-Atail) is normally added to a messenger RNA (mRNA) molecules to increasethe stability of the molecule. Immediately after transcription, the 3′end of the transcript is cleaved to free a 3′ hydroxyl. Then poly-Apolymerase adds a chain of adenosine nucleotides to the RNA. Theprocess, called polyadenylation, adds a poly-A tail that is between 100and 250 residues long.

Methods for the stabilization of RNA by incorporation ofchain-terminating nucleosides at the 3′-terminus include those describedin International Patent Publication No. WO2013103659, incorporatedherein in its entirety.

Unique poly-A tail lengths may provide certain advantages to thealternative RNAs of the present invention.

Generally, the length of a poly-A tail of the present invention isgreater than 10 nucleotides. In some embodiments, the poly-A tail isgreater than 20 nucleotides. In some embodiments, the poly-A tail isgreater than 30 nucleotides in length. In another embodiment, the poly-Atail is greater than 35 nucleotides in length. In another embodiment,the length of the poly-A tail is at least 40 nucleotides. In anotherembodiment, the length of the poly-A tail is at least 45 nucleotides. Inanother embodiment, the length of the poly-A tail is at least 55nucleotides. In another embodiment, the length of the poly-A tail is atleast 60 nucleotides. In another embodiment, the length of the poly-Atail is at least 70 nucleotides. In another embodiment, the length ofthe poly-A tail is at least 80 nucleotides. In another embodiment, thelength of the poly-A tail is at least 90 nucleotides. In anotherembodiment, the length of the poly-A tail is at least 100 nucleotides.In another embodiment, the length of the poly-A tail is at least 120nucleotides. In another embodiment, the length of the poly-A tail is atleast 140 nucleotides. In another embodiment, the length of the poly-Atail is at least 160 nucleotides. In another embodiment, the length ofthe poly-A tail is at least 180 nucleotides. In another embodiment, thelength of the poly-A tail is at least 200 nucleotides. In anotherembodiment, the length of the poly-A tail is at least 250 nucleotides.In another embodiment, the length of the poly-A tail is at least 300nucleotides.

In another embodiment, the length of the mRNA is at least 350nucleotides. In another embodiment, the length of the mRNA is at least400 nucleotides. In another embodiment, the length of the mRNA is atleast 450 nucleotides. In another embodiment, the length of the mRNA isat least 500 nucleotides. In another embodiment, the length of the mRNAis at least 600 nucleotides. In another embodiment, the length of themRNA is at least 700 nucleotides. In another embodiment, the length ofthe mRNA is at least 800 nucleotides. In another embodiment, the lengthof the mRNA is at least 900 nucleotides. In another embodiment, thelength of the mRNA is at least 1000 nucleotides. In another embodiment,the length of the mRNA is at least 1100 nucleotides. In anotherembodiment, the length of the mRNA is at least 1200 nucleotides. Inanother embodiment, the length of the mRNA is at least 1300 nucleotides.In another embodiment, the length of the mRNA is at least 1400nucleotides. In another embodiment, the length of the mRNA is at least1500 nucleotides. In another embodiment, the length of the mRNA is atleast 1600 nucleotides. In another embodiment, the length of the mRNA isat least 1700 nucleotides. In another embodiment, the length of the mRNAis at least 1800 nucleotides. In another embodiment, the length of themRNA is at least 1900 nucleotides. In another embodiment, the length ofthe mRNA is at least 2000 nucleotides. In another embodiment, the lengthof the mRNA is at least 2500 nucleotides. In another embodiment, thelength of the mRNA is at least 3000 nucleotides.

In one embodiment, the poly-A tail may be 80 nucleotides, 120nucleotides, 160 nucleotides in length on an alternative RNA moleculedescribed herein.

In another embodiment, the poly-A tail may be 20, 40, 80, 100, 120, 140or 160 nucleotides in length on an alternative RNA molecule describedherein.

In one embodiment, the poly-A tail is designed relative to the length ofthe overall alternative RNA molecule. This design may be based on thelength of the coding region of the alternative RNA, the length of aparticular feature or region of the alternative RNA (such as the mRNA),or based on the length of the ultimate product expressed from thealternative RNA. When relative to any additional feature of thealternative RNA (e.g., other than the mRNA portion which includes thepoly-A tail) the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90or 100% greater in length than the additional feature. The poly-A tailmay also be designed as a fraction of the alternative RNA to which itbelongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60,70, 80, or 90% or more of the total length of the construct or the totallength of the construct minus the poly-A tail.

In one embodiment, engineered binding sites and/or the conjugation ofnucleic acids or mRNA for Poly-A binding protein may be used to enhanceexpression. The engineered binding sites may be sensor sequences whichcan operate as binding sites for ligands of the local microenvironmentof the nucleic acids and/or mRNA. As a non-limiting example, the nucleicacids and/or mRNA may comprise at least one engineered binding site toalter the binding affinity of Poly-A binding protein (PABP) and analogsthereof. The incorporation of at least one engineered binding site mayincrease the binding affinity of the PABP and analogs thereof.

Additionally, multiple distinct nucleic acids or mRNA may be linkedtogether to the PABP (Poly-A binding protein) through the 3′-end usingalternative nucleotides at the 3′-terminus of the poly-A tail.Transfection experiments can be conducted in relevant cell lines at andprotein production can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hrand day 7 post-transfection. As a non-limiting example, the transfectionexperiments may be used to evaluate the effect on PABP or analogsthereof binding affinity as a result of the addition of at least oneengineered binding site.

In one embodiment, a polyA tail may be used to modulate translationinitiation. While not wishing to be bound by theory, the polyA tailrecruits PABP which in turn can interact with translation initiationcomplex and thus may be essential for protein synthesis.

In another embodiment, a polyA tail may also be used in the presentinvention to protect against 3′-5′ exonuclease digestion.

In one embodiment, the nucleic acids or mRNA of the present inventionare designed to include a polyA-G Quartet. The G-quartet is a cyclichydrogen bonded array of four guanosine nucleotides that can be formedby G-rich sequences in both DNA and RNA. In this embodiment, theG-quartet is incorporated at the end of the poly-A tail. The resultantnucleic acid or mRNA may be assayed for stability, protein productionand other parameters including half-life at various time points. It hasbeen discovered that the polyA-G quartet results in protein productionequivalent to at least 75% of that seen using a poly-A tail of 120nucleotides alone.

In one embodiment, the nucleic acids or mRNA of the present inventionmay comprise a polyA tail and may be stabilized by the addition of achain terminating nucleoside. The nucleic acids and/or mRNA with a polyAtail may further comprise a 5′-cap structure.

In another embodiment, the nucleic acids or mRNA of the presentinvention may comprise a polyA-G Quartet. The nucleic acids and/or mRNAwith a polyA-G Quartet may further comprise a 5′-cap structure.

In one embodiment, the chain terminating nucleoside which may be used tostabilize the nucleic acid or mRNA comprising a polyA tail or polyA-GQuartet may be, but is not limited to, those described in InternationalPatent Publication No. WO2013103659, incorporated herein by reference inits entirety. In another embodiment, the chain terminating nucleosideswhich may be used with the present invention includes, but is notlimited to, 3′-deoxyadenosine (cordycepin), 3′-deoxyuridine,3′-deoxycytidine, 3′-deoxyguanosine, 3′-deoxythymidine,2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine,2′,3′-dideoxyuridine, 2′,3′-dideoxycytidine, 2′,3′-dideoxyguanosine,2′,3′-dideoxythymidine, a 2′-deoxynucleoside, or a 2′ or3′-O-methylnucleoside.

In another embodiment, the nucleic acid such as, but not limited tomRNA, which comprise a polyA tail or a polyA-G Quartet may be stabilizedby an alteration to the 3′-region of the nucleic acid that can preventand/or inhibit the addition of oligio(U) (see e.g., International PatentPublication No. WO2013103659, incorporated herein by reference in itsentirety).

In yet another embodiment, the nucleic acid such as, but not limited tomRNA, which comprise a polyA tail or a polyA-G Quartet may be stabilizedby the addition of an oligonucleotide that terminates in a3′-deoxynucleoside, 2′,3′-dideoxynucleoside 3′-O-methylnucleosides,3′-O-ethyl nucleosides, 3′-arabinosides, and other alternativenucleosides known in the art and/or described herein.

5′-UTR, 3′-UTR and Translation Enhancer Elements (TEEs)

In one embodiment, the 5′-UTR of the polynucleotides, primaryconstructs, alternative nucleic acids and/or mmRNA may include at leastone translational enhancer polynucleotide, translation enhancer element,translational enhancer elements (collectively referred to as “TEE” s).As a non-limiting example, the TEE may be located between thetranscription promoter and the start codon. The polynucleotides, primaryconstructs, alternative nucleic acids and/or mmRNA with at least one TEEin the 5′-UTR may include a cap at the 5′-UTR. Further, at least one TEEmay be located in the 5′-UTR of polynucleotides, primary constructs,alternative nucleic acids and/or mmRNA undergoing cap-dependent orcap-independent translation.

The term “translational enhancer element” or “translation enhancerelement” (herein collectively referred to as “TEE”) refers to sequencesthat increase the amount of polypeptide or protein produced from anmRNA.

In one aspect, TEEs are conserved elements in the UTR which can promotetranslational activity of a nucleic acid such as, but not limited to,cap-dependent or cap-independent translation. The conservation of thesesequences has been previously shown by Panek et al (Nucleic AcidsResearch, 2013, 1-10; incorporated herein by reference in its entirety)across 14 species including humans.

In one non-limiting example, the TEEs known may be in the 5′-leader ofthe Gtx homeodomain protein (Chappell et al., Proc. Natl. Acad. Sci. USA101:9590-9594, 2004, incorporated herein by reference in theirentirety).

In another non-limiting example, TEEs are disclosed as SEQ ID NOs: 1-35in US Patent Publication No. US20090226470, SEQ ID NOs: 1-35 in USPatent Publication US20130177581, SEQ ID NOs: 1-35 in InternationalPatent Publication No. WO2009075886, SEQ ID NOs: 1-5, and 7-645 inInternational Patent Publication No. WO2012009644, SEQ ID NO: 1 inInternational Patent Publication No. WO1999024595, SEQ ID NO: 1 in U.S.Pat. No. 6,310,197, and SEQ ID NO: 1 in U.S. Pat. No. 6,849,405, each ofwhich is incorporated herein by reference in its entirety.

In yet another non-limiting example, the TEE may be an internal ribosomeentry site (IRES), HCV-IRES or an IRES element such as, but not limitedto, those described in U.S. Pat. No. 7,468,275, US Patent PublicationNos. US20070048776 and US20110124100 and International PatentPublication Nos. WO2007025008 and WO2001055369, each of which isincorporated herein by reference in its entirety. The IRES elements mayinclude, but are not limited to, the Gtx sequences (e.g., Gtx9-nt,Gtx8-nt, Gtx7-nt) described by Chappell et al. (Proc. Natl. Acad. Sci.USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278, 2005) andin US Patent Publication Nos. US20070048776 and US20110124100 andInternational Patent Publication No. WO2007025008, each of which isincorporated herein by reference in its entirety.

“Translational enhancer polynucleotides” or “translation enhancerpolynucleotide sequences” are polynucleotides which include one or moreof the specific TEE exemplified herein and/or disclosed in the art (seee.g., U.S. Pat. No. 6,310,197, U.S. Pat. No. 6,849,405, U.S. Pat. No.7,456,273, U.S. Pat. No. 7,183,395, US20090226470, US20070048776,US20110124100, US20090093049, US20130177581, WO2009075886, WO2007025008,WO2012009644, WO2001055371 WO1999024595, and EP2610341A1 andEP2610340A1; each of which is incorporated herein by reference in itsentirety) or their variants, homologs or functional derivatives. One ormultiple copies of a specific TEE can be present in the polynucleotides,primary constructs, alternative nucleic acids and/or mm RNA. The TEEs inthe translational enhancer polynucleotides can be organized in one ormore sequence segments. A sequence segment can harbor one or more of thespecific TEEs exemplified herein, with each TEE being present in one ormore copies. When multiple sequence segments are present in atranslational enhancer polynucleotide, they can be homogenous orheterogeneous. Thus, the multiple sequence segments in a translationalenhancer polynucleotide can harbor identical or different types of thespecific TEEs exemplified herein, identical or different number ofcopies of each of the specific TEEs, and/or identical or differentorganization of the TEEs within each sequence segment.

In one embodiment, the polynucleotides, primary constructs, alternativenucleic acids and/or mmRNA may include at least one TEE that isdescribed in International Patent Publication No. WO1999024595,WO2012009644, WO2009075886, WO2007025008, WO1999024595, European PatentPublication No. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197,U.S. Pat. No. 6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No.7,183,395, US Patent Publication No. US20090226470, US20110124100,US20070048776, US20090093049, and US20130177581 each of which isincorporated herein by reference in its entirety. The TEE may be locatedin the 5′-UTR of the polynucleotides, primary constructs, alternativenucleic acids and/or mmRNA.

In another embodiment, the polynucleotides, primary constructs,alternative nucleic acids and/or mmRNA may include at least one TEE thathas at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95% or at least 99% identity with the TEEs described in US PatentPublication Nos. US20090226470, US20070048776, US20130177581 andUS20110124100, International Patent Publication No. WO1999024595,WO2012009644, WO2009075886 and WO2007025008, European Patent PublicationNo. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.6,849,405, U.S. Pat. No. 7,456,273, U.S. Pat. No. 7,183,395, each ofwhich is incorporated herein by reference in its entirety.

In one embodiment, the 5′-UTR of the polynucleotides, primaryconstructs, alternative nucleic acids and/or mmRNA may include at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18 at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 55 or more than 60 TEE sequences. The TEEsequences in the 5′-UTR of the polynucleotides, primary constructs,alternative nucleic acids and/or mmRNA of the present invention may bethe same or different TEE sequences. The TEE sequences may be in apattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereofrepeated once, twice, or more than three times. In these patterns, eachletter, A, B, or C represent a different TEE sequence at the nucleotidelevel.

In one embodiment, the 5′-UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 5′-UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times and atleast 9 times or more than 9 times in the 5′-UTR.

In another embodiment, the spacer separating two TEE sequences mayinclude other sequences known in the art which may regulate thetranslation of the polynucleotides, primary constructs, alternativenucleic acids and/or mmRNA of the present invention such as, but notlimited to, miR sequences described herein (e.g., miR binding sites andmiR seeds). As a non-limiting example, each spacer used to separate twoTEE sequences may include a different miR sequence or component of a miRsequence (e.g., miR seed sequence).

In one embodiment, the TEE in the 5′-UTR of the polynucleotides, primaryconstructs, alternative nucleic acids and/or mmRNA of the presentinvention may include at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99% or more than 99% of the TEE sequences disclosed in USPatent Publication Nos. US20090226470, US20070048776, US20130177581 andUS20110124100, International Patent Publication No. WO1999024595,WO2012009644, WO2009075886 and WO2007025008, European Patent PublicationNo. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.6,849,405, U.S. Pat. No. 7,456,273, and U.S. Pat. No. 7,183,395 each ofwhich is incorporated herein by reference in its entirety. In anotherembodiment, the TEE in the 5′-UTR of the polynucleotides, primaryconstructs, alternative nucleic acids and/or mmRNA of the presentinvention may include a 5-30 nucleotide fragment, a 5-25 nucleotidefragment, a 5-20 nucleotide fragment, a 5-15 nucleotide fragment, a 5-10nucleotide fragment of the TEE sequences disclosed in US PatentPublication Nos. US20090226470, US20070048776, US20130177581 andU520110124100, International Patent Publication No. WO1999024595,WO2012009644, WO2009075886 and WO2007025008, European Patent PublicationNo. EP2610341A1 and EP2610340A1, U.S. Pat. No. 6,310,197, U.S. Pat. No.6,849,405, U.S. Pat. No. 7,456,273, and U.S. Pat. No. 7,183,395; each ofwhich is incorporated herein by reference in its entirety.

In one embodiment, the TEE in the 5′-UTR of the polynucleotides, primaryconstructs, alternative nucleic acids and/or mmRNA of the presentinvention may include at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 99% or more than 99% of the TEE sequences disclosed inChappell et al. (Proc. Natl. Acad. Sci. USA 101:9590-9594, 2004) andZhou et al. (PNAS 102:6273-6278, 2005), in Supplemental Table 1 and inSupplemental Table 2 disclosed by Wellensiek et al (Genome-wideprofiling of human cap-independent translation-enhancing elements,Nature Methods, 2013; DOI:10.1038/NMETH.2522); each of which is hereinincorporated by reference in its entirety. In another embodiment, theTEE in the 5′-UTR of the polynucleotides, primary constructs,alternative nucleic acids and/or mmRNA of the present invention mayinclude a 5-30 nucleotide fragment, a 5-25 nucleotide fragment, a 5-20nucleotide fragment, a 5-15 nucleotide fragment, a 5-10 nucleotidefragment of the TEE sequences disclosed in Chappell et al. (Proc. Natl.Acad. Sci. USA 101:9590-9594, 2004) and Zhou et al. (PNAS 102:6273-6278,2005), in Supplemental Table 1 and in Supplemental Table 2 disclosed byWellensiek et al (Genome-wide profiling of human cap-independenttranslation-enhancing elements, Nature Methods, 2013;DOI:10.1038/NMETH.2522); each of which is incorporated herein byreference in its entirety.

In one embodiment, the TEE used in the 5′-UTR of the polynucleotides,primary constructs, alternative nucleic acids and/or mm RNA of thepresent invention is an IRES sequence such as, but not limited to, thosedescribed in U.S. Pat. No. 7,468,275 and International PatentPublication No. WO2001055369, each of which is incorporated herein byreference in its entirety.

In one embodiment, the TEEs used in the 5′-UTR of the polynucleotides,primary constructs, alternative nucleic acids and/or mm RNA of thepresent invention may be identified by the methods described in USPatent Publication No. US20070048776 and US20110124100 and InternationalPatent Publication Nos. WO2007025008 and WO2012009644, each of which isincorporated herein by reference in its entirety.

In another embodiment, the TEEs used in the 5′-UTR of thepolynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present invention may be a transcription regulatory elementdescribed in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, USPatent Publication No. US20090093049, and International Publication No.WO2001055371, each of which is incorporated herein by reference in itsentirety. The transcription regulatory elements may be identified bymethods known in the art, such as, but not limited to, the methodsdescribed in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, USPatent Publication No. US20090093049, and International Publication No.WO2001055371, each of which is incorporated herein by reference in itsentirety.

In yet another embodiment, the TEE used in the 5′-UTR of thepolynucleotides, primary constructs, alternative nucleic acids and/or mmRNA of the present invention is an oligonucleotide or portion thereof asdescribed in U.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, USPatent Publication No. US20090093049, and International Publication No.WO2001055371, each of which is incorporated herein by reference in itsentirety.

The 5′ UTR comprising at least one TEE described herein may beincorporated in a monocistronic sequence such as, but not limited to, avector system or a nucleic acid vector. As a non-limiting example, thevector systems and nucleic acid vectors may include those described inU.S. Pat. No. 7,456,273 and U.S. Pat. No. 7,183,395, US PatentPublication No. US20070048776, US20090093049 and US20110124100 andInternational Patent Publication Nos. WO2007025008 and WO2001055371,each of which is incorporated herein by reference in its entirety.

In one embodiment, the TEEs described herein may be located in the5′-UTR and/or the 3′-UTR of the polynucleotides, primary constructs,alternative nucleic acids and/or mmRNA. The TEEs located in the 3′-UTRmay be the same and/or different than the TEEs located in and/ordescribed for incorporation in the 5′-UTR.

In one embodiment, the 3′-UTR of the polynucleotides, primaryconstructs, alternative nucleic acids and/or mmRNA may include at least1, at least 2, at least 3, at least 4, at least 5, at least 6, at least7, at least 8, at least 9, at least 10, at least 11, at least 12, atleast 13, at least 14, at least 15, at least 16, at least 17, at least18 at least 19, at least 20, at least 21, at least 22, at least 23, atleast 24, at least 25, at least 30, at least 35, at least 40, at least45, at least 50, at least 55 or more than 60 TEE sequences. The TEEsequences in the 3′-UTR of the polynucleotides, primary constructs,alternative nucleic acids and/or mmRNA of the present invention may bethe same or different TEE sequences. The TEE sequences may be in apattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereofrepeated once, twice, or more than three times. In these patterns, eachletter, A, B, or C represent a different TEE sequence at the nucleotidelevel.

In one embodiment, the 3′-UTR may include a spacer to separate two TEEsequences. As a non-limiting example, the spacer may be a 15 nucleotidespacer and/or other spacers known in the art. As another non-limitingexample, the 3′-UTR may include a TEE sequence-spacer module repeated atleast once, at least twice, at least 3 times, at least 4 times, at least5 times, at least 6 times, at least 7 times, at least 8 times and atleast 9 times or more than 9 times in the 3′-UTR.

In another embodiment, the spacer separating two TEE sequences mayinclude other sequences known in the art which may regulate thetranslation of the polynucleotides, primary constructs, alternativenucleic acids and/or mmRNA of the present invention such as, but notlimited to, miR sequences described herein (e.g., miR binding sites andmiR seeds). As a non-limiting example, each spacer used to separate twoTEE sequences may include a different miR sequence or component of a miRsequence (e.g., miR seed sequence).

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′-UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, herein incorporated byreference in its entirety).

Heterologous 5′-UTRs

A 5′ UTR may be provided as a flanking region to the alternative nucleicacids (mRNA), enhanced alternative RNA or ribonucleic acids of theinvention. 5′-UTR may be homologous or heterologous to the coding regionfound in the alternative nucleic acids (mRNA), enhanced alternative RNAor ribonucleic acids of the invention. Multiple 5′ UTRs may be includedin the flanking region and may be the same or of different sequences.Any portion of the flanking regions, including none, may be codonoptimized and any may independently contain one or more differentstructural or chemical alterations, before and/or after codonoptimization.

Shown in Lengthy Table 21 in U.S. Provisional Application No.61/775,509, and in Lengthy Table 21 and in Table 22 in U.S. ProvisionalApplication No. 61/829,372, the contents of each of which areincorporated herein by reference in their entirety, is a listing of thestart and stop site of the alternative nucleic acids (mRNA), enhancedalternative RNA or ribonucleic acids of the invention. In Table 21 each5′-UTR (5′-UTR-005 to 5′-UTR 68511) is identified by its start and stopsite relative to its native or wild-type (homologous) transcript (ENST;the identifier used in the ENSEMBL database).

To alter one or more properties of the polynucleotides, primaryconstructs or mmRNA of the invention, 5′-UTRs which are heterologous tothe coding region of the alternative nucleic acids (mRNA), enhancedalternative RNA or ribonucleic acids of the invention are engineeredinto compounds of the invention. The alternative nucleic acids (mRNA),enhanced alternative RNA or ribonucleic acids are then administered tocells, tissue or organisms and outcomes such as protein level,localization and/or half-life are measured to evaluate the beneficialeffects the heterologous 5′-UTR may have on the alternative nucleicacids (mRNA), enhanced alternative RNA or ribonucleic acids of theinvention. Variants of the 5′ UTRs may be utilized wherein one or morenucleotides are added or removed to the termini, including A, T, C or G.5′-UTRs may also be codon-optimized or altered in any manner describedherein.

Incorporating microRNA Binding Sites

In one embodiment, alternative nucleic acids (mRNA), enhancedalternative RNA or ribonucleic acids of the invention would not onlyencode a polypeptide but also a sensor sequence. Sensor sequencesinclude, for example, microRNA binding sites, transcription factorbinding sites, structured mRNA sequences and/or motifs, artificialbinding sites engineered to act as pseudo-receptors for endogenousnucleic acid binding molecules. Non-limiting examples, ofpolynucleotides comprising at least one sensor sequence are described inco-pending and co-owned U.S. Provisional Patent Application No.61/753,661, filed Jan. 17, 2013, U.S. Provisional Patent Application No.61/754,159, filed Jan. 18, 2013, U.S. Provisional Patent Application No.61/781,097, filed Mar. 14, 2013, U.S. Provisional Patent Application No.61/829,334, filed May 31, 2013, U.S. Provisional Patent Application No.61/839,893, filed Jun. 27, 2013, U.S. Provisional Patent Application No.61/842,733, filed Jul. 3, 2013, and U.S. Provisional Patent ApplicationNo. 61/857,304, filed Jul. 23, 2013, the contents of each of which areincorporated herein by reference in their entirety.

In one embodiment, microRNA (miRNA) profiling of the target cells ortissues is conducted to determine the presence or absence of miRNA inthe cells or tissues.

microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bindto the 3′UTR of nucleic acid molecules and down-regulate gene expressioneither by reducing nucleic acid molecule stability or by inhibitingtranslation. The alternative nucleic acids (mRNA), enhanced alternativeRNA or ribonucleic acids of the invention may comprise one or moremicroRNA target sequences, microRNA sequences, or microRNA seeds. Suchsequences may correspond to any known microRNA such as those taught inUS Publication US2005/0261218 and US Publication US2005/0059005, thecontents of which are incorporated herein by reference in theirentirety.

A microRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature microRNA, which sequence hasperfect Watson-Crick complementarity to the miRNA target sequence. AmicroRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA.In some embodiments, a microRNA seed may comprise 7 nucleotides (e.g.,nucleotides 2-8 of the mature microRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenosine (A)opposed to microRNA position 1. In some embodiments, a microRNA seed maycomprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA),wherein the seed-complementary site in the corresponding miRNA target isflanked by an adenosine (A) opposed to microRNA position 1. See forexample, Grimson A, Farh K K, Johnston W K, Garrett-Engele P, Lim L P,Bartel D P; Mol Cell. 2007 Jul. 6; 27(1):91-105. The bases of themicroRNA seed have complete complementarity with the target sequence. Byengineering microRNA target sequences into the 3′UTR of nucleic acids ormRNA of the invention one can target the molecule for degradation orreduced translation, provided the microRNA in question is available.This process will reduce the hazard of off target effects upon nucleicacid molecule delivery. Identification of microRNA, microRNA targetregions, and their expression patterns and role in biology have beenreported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand andCheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia2012 26:404-413 (2011 Dec. 20. doi: 10.1038/Ieu.2011.356); Bartel Cell2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393-403 and all references therein;each of which is incorporated herein by reference in its entirety).

For example, if the mRNA is not intended to be delivered to the liverbut ends up there, then miR-122, a microRNA abundant in liver, caninhibit the expression of the gene of interest if one or multiple targetsites of miR-122 are engineered into the 3′UTR of the alternativenucleic acids, enhanced alternative RNA or ribonucleic acids.Introduction of one or multiple binding sites for different microRNA canbe engineered to further decrease the longevity, stability, and proteintranslation of an alternative nucleic acids, enhanced alternative RNA orribonucleic acids. As used herein, the term “microRNA site” refers to amicroRNA target site or a microRNA recognition site, or any nucleotidesequence to which a microRNA binds or associates. It should beunderstood that “binding” may follow traditional Watson-Crickhybridization rules or may reflect any stable association of themicroRNA with the target sequence at or adjacent to the microRNA site.

Conversely, for the purposes of the alternative nucleic acids, enhancedalternative RNA or ribonucleic acids of the present invention, microRNAbinding sites can be engineered out of (i.e. removed from) sequences inwhich they naturally occur in order to increase protein expression inspecific tissues. For example, miR-122 binding sites may be removed toimprove protein expression in the liver.

In one embodiment, the alternative nucleic acids, enhanced alternativeRNA or ribonucleic acids of the present invention may include at leastone miRNA-binding site in the 3′-UTR in order to direct cytotoxic orcytoprotective mRNA therapeutics to specific cells such as, but notlimited to, normal and/or cancerous cells (e.g., HEP3B or SNU449).

In another embodiment, the alternative nucleic acids, enhancedalternative RNA or ribonucleic acids of the present invention mayinclude three miRNA-binding sites in the 3′-UTR in order to directcytotoxic or cytoprotective mRNA therapeutics to specific cells such as,but not limited to, normal and/or cancerous cells (e.g., HEP3B orSNU449).

Regulation of expression in multiple tissues can be accomplished throughintroduction or removal or one or several microRNA binding sites. Thedecision of removal or insertion of microRNA binding sites, or anycombination, is dependent on microRNA expression patterns and theirprofilings in diseases.

Examples of tissues where microRNA are known to regulate mRNA, andthereby protein expression, include, but are not limited to, liver(miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells(miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16,miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126).

Specifically, microRNAs are known to be differentially expressed inimmune cells (also called hematopoietic cells), such as antigenpresenting cells (APCs) (e.g. dendritic cells and macrophages),macrophages, monocytes, B lymphocytes, T lymphocytes, granuocytes,natural killer cells, etc. Immune cell specific microRNAs are involvedin immunogenicity, autoimmunity, the immune-response to infection,inflammation, as well as unwanted immune response after gene therapy andtissue/organ transplantation. Immune cells specific microRNAs alsoregulate many aspects of development, proliferation, differentiation andapoptosis of hematopoietic cells (immune cells). For example, miR-142and miR-146 are exclusively expressed in the immune cells, particularlyabundant in myeloid dendritic cells. It was demonstrated in the art thatthe immune response to exogenous nucleic acid molecules was shut-off byadding miR-142 binding sites to the 3′-UTR of the delivered geneconstruct, enabling more stable gene transfer in tissues and cells.miR-142 efficiently degrades the exogenous mRNA in antigen presentingcells and suppresses cytotoxic elimination of transduced cells (Annoni Aet al., blood, 2009, 114, 5152-5161; Brown B D, et al., Nat med. 2006,12(5), 585-591; Brown B D, et al., blood, 2007, 110(13): 4144-4152, eachof which is incorporated herein by reference in its entirety).

An antigen-mediated immune response can refer to an immune responsetriggered by foreign antigens, which, when entering an organism, areprocessed by the antigen presenting cells and displayed on the surfaceof the antigen presenting cells. T cells can recognize the presentedantigen and induce a cytotoxic elimination of cells that express theantigen.

Introducing the miR-142 binding site into the 3′-UTR of a polypeptide ofthe present invention can selectively repress the gene expression in theantigen presenting cells through miR-142 mediated mRNA degradation,limiting antigen presentation in APCs (e.g. dendritic cells) and therebypreventing antigen-mediated immune response after the delivery of thepolynucleotides. The polynucleotides are therefore stably expressed intarget tissues or cells without triggering cytotoxic elimination.

In one embodiment, microRNAs binding sites that are known to beexpressed in immune cells, in particular, the antigen presenting cells,can be engineered into the polynucleotide to suppress the expression ofthe sensor-signal polynucleotide in APCs through microRNA mediated RNAdegradation, subduing the antigen-mediated immune response, while theexpression of the polynucleotide is maintained in non-immune cells wherethe immune cell specific microRNAs are not expressed. For example, toprevent the immunogenic reaction caused by a liver specific proteinexpression, the miR-122 binding site can be removed and the miR-142(and/or mirR-146) binding sites can be engineered into the 3-UTR of thepolynucleotide.

To further drive the selective degradation and suppression of mRNA inAPCs and macrophage, the polynucleotide may include another negativeregulatory element in the 3-UTR, either alone or in combination withmir-142 and/or mir-146 binding sites. As a non-limiting example, oneregulatory element is the Constitutive Decay Elements (CDEs).

Immune cells specific microRNAs include, but are not limited to,hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p,hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p,hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p,hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p,miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p,miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p,miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a-5p,miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p,miR-15a-3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p,miR-16-2-3p, miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p,miR-181a-2-3p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p,miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p, miR-27b-5p,miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-1-5p,miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p, miR-30e-5p,miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346,miR-34a-3p, miR-34a-5p, miR-363-3p, miR-363-5p, miR-372, miR-377-3p,miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p,miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935,miR-99a-3p, miR-99a-5p, miR-99b-3p and miR-99b-5p. Furthermore, novelmiroRNAs are discovered in the immune cells in the art throughmicro-array hybridization and microtome analysis (Jima D D et al, Blood,2010, 116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, thecontent of each of which is incorporated herein by reference in itsentirety.)

MicroRNAs that are known to be expressed in the liver include, but arenot limited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p,miR-1228-5p, miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p,miR-152, miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p,miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, miR-939-5p.MicroRNA binding sites from any liver specific microRNA can beintroduced to or removed from the polynucleotides to regulate theexpression of the polynucleotides in the liver. Liver specific microRNAsbinding sites can be engineered alone or further in combination withimmune cells (e.g. APCs) microRNA binding sites in order to preventimmune reaction against protein expression in the liver.

MicroRNAs that are known to be expressed in the lung include, but arenot limited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p,miR-126-5p, miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p,miR-130b-3p, miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p,miR-18a-5p, miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p,miR-296-3p, miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p,miR-381-5p. MicroRNA binding sites from any lung specific microRNA canbe introduced to or removed from the polynucleotide to regulate theexpression of the polynucleotide in the lung. Lung specific microRNAsbinding sites can be engineered alone or further in combination withimmune cells (e.g. APCs) microRNA binding sites in order to prevent animmune reaction against protein expression in the lung.

MicroRNAs that are known to be expressed in the heart include, but arenot limited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p,miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p,miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p,miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p and miR-92b-5p. MicroRNAbinding sites from any heart specific microRNA can be introduced to orremoved from the polynucleotides to regulate the expression of thepolynucleotides in the heart. Heart specific microRNAs binding sites canbe engineered alone or further in combination with immune cells (e.g.APCs) microRNA binding sites to prevent an immune reaction againstprotein expression in the heart.

MicroRNAs that are known to be expressed in the nervous system include,but are not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p,miR-125b-1-3p, miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p,miR-128, miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p,miR-137, miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153,miR-181c-3p, miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b,miR-212-3p, miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p,miR-23a-5p, miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p,miR-30c-2-3p, miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329, miR-342-3p,miR-3665, miR-3666, miR-380-3p, miR-380-5p, miR-383, miR-410,miR-425-3p, miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510,miR-516a-3p, miR-548b-5p, miR-548c-5p, miR-571, miR-7-1-3p, miR-7-2-3p,miR-7-5p, miR-802, miR-922, miR-9-3p and miR-9-5p. MicroRNAs enriched inthe nervous system further include those specifically expressed inneurons, including, but not limited to, miR-132-3p, miR-132-3p,miR-148b-3p, miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p,miR-212-5p, miR-320b, miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p,miR-325, miR-326, miR-328, miR-922 and those specifically expressed inglial cells, including, but not limited to, miR-1250, miR-219-1-3p,miR-219-2-3p, miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p,miR-3065-5p, miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, miR-657.MicroRNA binding sites from any CNS specific microRNA can be introducedto or removed from the polynucleotides to regulate the expression of thepolynucleotide in the nervous system. Nervous system specific microRNAsbinding sites can be engineered alone or further in combination withimmune cells (e.g. APCs) microRNA binding sites in order to preventimmune reaction against protein expression in the nervous system.

MicroRNAs that are known to be expressed in the pancreas include, butare not limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p,miR-195-5p, miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p,miR-216a-3p, miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375,miR-7-1-3p, miR-7-2-3p, miR-493-3p, miR-493-5p and miR-944. MicroRNAbinding sites from any pancreas specific microRNA can be introduced toor removed from the polynucleotide to regulate the expression of thepolynucleotide in the pancreas. Pancreas specific microRNAs bindingsites can be engineered alone or further in combination with immunecells (e.g. APCs) microRNA binding sites in order to prevent an immunereaction against protein expression in the pancreas.

MicroRNAs that are known to be expressed in the kidney further include,but are not limited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p,miR-192-5p, miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p,miR-204-5p, miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p,miR30c-5p, miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5pand miR-562. MicroRNA binding sites from any kidney specific microRNAcan be introduced to or removed from the polynucleotide to regulate theexpression of the polynucleotide in the kidney. Kidney specificmicroRNAs binding sites can be engineered alone or further incombination with immune cells (e.g. APCs) microRNA binding sites toprevent an immune reaction against protein expression in the kidney.

MicroRNAs that are known to be expressed in the muscle further include,but are not limited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a,miR-133b, miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p,miR-188-3p, miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p andmiR-25-5p. MicroRNA binding sites from any muscle specific microRNA canbe introduced to or removed from the polynucleotide to regulate theexpression of the polynucleotide in the muscle. Muscle specificmicroRNAs binding sites can be engineered alone or further incombination with immune cells (e.g. APCs) microRNA binding sites toprevent an immune reaction against protein expression in the muscle.

MicroRNAs are differentially expressed in different types of cells, suchas endothelial cells, epithelial cells and adipocytes. For example,microRNAs that are expressed in endothelial cells include, but are notlimited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p, miR-101-3p,miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p, miR-1236-5p,miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p, miR-18a-5p,miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p, miR-19b-3p,miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p, miR-21-5p,miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p, miR-23a-5p,miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p, miR-424-5p,miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p, miR-92b-3p andmiR-92b-5p. Many novel microRNAs are discovered in endothelial cellsfrom deep-sequencing analysis (Voellenkle C et al., RNA, 2012, 18,472-484, herein incorporated by reference in its entirety) microRNAbinding sites from any endothelial cell specific microRNA can beintroduced to or removed from the polynucleotide to modulate theexpression of the polynucleotide in the endothelial cells in variousconditions.

For further example, microRNAs that are expressed in epithelial cellsinclude, but are not limited to, let-7b-3p, let-7b-5p, miR-1246,miR-200a-3p, miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p,miR-200c-5p, miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802and miR-34a, miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5pspecific in respiratory ciliated epithelial cells; let-7 family,miR-133a, miR-133b, miR-126 specific in lung epithelial cells;miR-382-3p, miR-382-5p specific in renal epithelial cells and miR-762specific in corneal epithelial cells. MicroRNA binding sites from anyepithelial cell specific MicroRNA can be introduced to or removed fromthe polynucleotide to modulate the expression of the polynucleotide inthe epithelial cells in various conditions.

In addition, a large group of microRNAs are enriched in embryonic stemcells, controlling stem cell self-renewal as well as the developmentand/or differentiation of various cell lineages, such as neural cells,cardiac, hematopoietic cells, skin cells, osteogenic cells and musclecells (Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764;Vidigal J A and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436;Goff L A et al., PLoS One, 2009, 4:e7192; Morin R D et al., Genome Res,2008, 18, 610-621; Yoo J K et al., Stem Cells Dev. 2012, 21(11),2049-2057, each of which is herein incorporated by reference in itsentirety). MicroRNAs abundant in embryonic stem cells include, but arenot limited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p,miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p,miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p,miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p,miR-423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f,miR-548g-3p, miR-548g-5p, miR-548i, miR-548k, miR-548l, miR-548m,miR-548n, miR-5480-3p, miR-5480-5p, miR-548p, miR-664a-3p, miR-664a-5p,miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p,miR-885-5p, miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p,miR-99b-3p and miR-99b-5p. Many predicted novel microRNAs are discoveredby deep sequencing in human embryonic stem cells (Morin R D et al.,Genome Res, 2008, 18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each ofwhich is incorporated herein by references in its entirety).

In one embodiment, the binding sites of embryonic stem cell specificmicroRNAs can be included in or removed from the 3-UTR of thepolynucleotide to modulate the development and/or differentiation ofembryonic stem cells, to inhibit the senescence of stem cells in adegenerative condition (e.g. degenerative diseases), or to stimulate thesenescence and apoptosis of stem cells in a disease condition (e.g.cancer stem cells).

Many microRNA expression studies are conducted in the art to profile thedifferential expression of microRNAs in various cancer cells/tissues andother diseases. Some microRNAs are abnormally over-expressed in certaincancer cells and others are under-expressed. For example, microRNAs aredifferentially expressed in cancer cells (WO2008/154098, US2013/0059015,US2013/0042333, WO2011/157294); cancer stem cells (US2012/0053224);pancreatic cancers and diseases (US2009/0131348, US2011/0171646,US2010/0286232, U.S. Pat. No. 8,389,210); asthma and inflammation (U.S.Pat. No. 8,415,096); prostate cancer (US2013/0053264); hepatocellularcarcinoma (WO2012/151212, US2012/0329672, WO2008/054828, U.S. Pat. No.8,252,538); lung cancer cells (WO2011/076143, WO2013/033640,WO2009/070653, US2010/0323357); cutaneous T cell lymphoma(WO2013/011378); colorectal cancer cells (WO2011/0281756,WO2011/076142); cancer positive lymph nodes (WO2009/100430,US2009/0263803); nasopharyngeal carcinoma (EP21 12235); chronicobstructive pulmonary disease (US2012/0264626, US2013/0053263); thyroidcancer (WO2013/066678); ovarian cancer cells US2012/0309645,WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974,US2012/0316081, US2012/0283310, WO2010/018563, the content of each ofwhich is incorporated herein by reference in its entirety.)

As a non-limiting example, microRNA sites that are over-expressed incertain cancer and/or tumor cells can be removed from the 3-UTR of thepolynucleotide encoding the polypeptide of interest, restoring theexpression suppressed by the over-expressed microRNAs in cancer cells,thus ameliorating the corresponsive biological function, for instance,transcription stimulation and/or repression, cell cycle arrest,apoptosis and cell death. Normal cells and tissues, wherein microRNAsexpression is not up-regulated, will remain unaffected.

MicroRNA can also regulate complex biological processes such asangiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the alternative nucleic acids, enhanced alternative RNAor ribonucleic acids of the invention, binding sites for microRNAs thatare involved in such processes may be removed or introduced, in order totailor the expression of the alternative nucleic acids, enhancedalternative RNA or ribonucleic acids expression to biologically relevantcell types or to the context of relevant biological processes. In thiscontext, the mRNA are defined as auxotrophic mRNA.

MicroRNA gene regulation may be influenced by the sequence surroundingthe microRNA such as, but not limited to, the species of the surroundingsequence, the type of sequence (e.g., heterologous, homologous andartificial), regulatory elements in the surrounding sequence and/orstructural elements in the surrounding sequence. The microRNA may beinfluenced by the 5′-UTR and/or the 3′-UTR. As a non-limiting example, anon-human 3′-UTR may increase the regulatory effect of the microRNAsequence on the expression of a polypeptide of interest compared to ahuman 3′-UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elementsof the 5′-UTR can influence microRNA mediated gene regulation. Oneexample of a regulatory element and/or structural element is astructured IRES (Internal Ribosome Entry Site) in the 5′-UTR, which isnecessary for the binding of translational elongation factors toinitiate protein translation. EIF4A2 binding to this secondarilystructured element in the 5′-UTR is necessary for microRNA mediated geneexpression (Meijer H A et al., Science, 2013, 340, 82-85, hereinincorporated by reference in its entirety). The alternative nucleicacids, enhanced alternative RNA or ribonucleic acids of the inventioncan further be alternative to include this structured 5′-UTR in order toenhance microRNA mediated gene regulation.

At least one microRNA site can be engineered into the 3′ UTR of thealternative nucleic acids, enhanced alternative RNA or ribonucleic acidsof the present invention. In this context, at least two, at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, at least ten or more microRNA sites may beengineered into the 3′ UTR of the ribonucleic acids of the presentinvention. In one embodiment, the microRNA sites incorporated into thealternative nucleic acids, enhanced alternative RNA or ribonucleic acidsmay be the same or may be different microRNA sites. In anotherembodiment, the microRNA sites incorporated into the alternative nucleicacids, enhanced alternative RNA or ribonucleic acids may target the sameor different tissues in the body. As a non-limiting example, through theintroduction of tissue-, cell-type-, or disease-specific microRNAbinding sites in the 3′ UTR of an alternative nucleic acid mRNA, thedegree of expression in specific cell types (e.g. hepatocytes, myeloidcells, endothelial cells, cancer cells, etc.) can be reduced.

In one embodiment, a microRNA site can be engineered near the 5′terminus of the 3′-UTR, about halfway between the 5′ terminus and3′terminus of the 3′-UTR and/or near the 3′terminus of the 3′-UTR. As anon-limiting example, a microRNA site may be engineered near the 5′terminus of the 3′-UTR and about halfway between the 5′ terminus and3′terminus of the 3′-UTR. As another non-limiting example, a microRNAsite may be engineered near the 3′terminus of the 3′-UTR and abouthalfway between the 5′ terminus and 3′terminus of the 3′-UTR. As yetanother non-limiting example, a microRNA site may be engineered near the5′ terminus of the 3′-UTR and near the 3′ terminus of the 3′-UTR.

In another embodiment, a 3′-UTR can comprise 4 microRNA sites. ThemicroRNA sites may be complete microRNA binding sites, microRNA seedsequences and/or microRNA binding site sequences without the seedsequence.

In one embodiment, a nucleic acid of the invention may be engineered toinclude at least one microRNA in order to dampen the antigenpresentation by antigen presenting cells. The microRNA may be thecomplete microRNA sequence, the microRNA seed sequence, the microRNAsequence without the seed or a combination thereof. As a non-limitingexample, the microRNA incorporated into the nucleic acid may be specificto the hematopoietic system. As another non-limiting example, themicroRNA incorporated into the nucleic acid of the invention to dampenantigen presentation is miR-142-3p.

In one embodiment, a nucleic acid may be engineered to include microRNAsites which are expressed in different tissues of a subject. As anon-limiting example, an alternative nucleic acid, enhanced alternativeRNA or ribonucleic acid of the present invention may be engineered toinclude miR-192 and miR-122 to regulate expression of the alternativenucleic acid, enhanced alternative RNA or ribonucleic acid in the liverand kidneys of a subject. In another embodiment, an alternative nucleicacid, enhanced alternative RNA or ribonucleic acid may be engineered toinclude more than one microRNA sites for the same tissue. For example,an alternative nucleic acid, enhanced alternative RNA or ribonucleicacid of the present invention may be engineered to include miR-17-92 andmiR-126 to regulate expression of the alternative nucleic acid, enhancedalternative RNA or ribonucleic acid in endothelial cells of a subject.

In one embodiment, the therapeutic window and or differential expressionassociated with the target polypeptide encoded by the alternativenucleic acid, enhanced alternative RNA or ribonucleic acid encoding asignal (also referred to herein as a polynucleotide) of the inventionmay be altered. For example, polynucleotides may be designed whereby adeath signal is more highly expressed in cancer cells (or a survivalsignal in a normal cell) by virtue of the miRNA signature of thosecells. Where a cancer cell expresses a lower level of a particularmiRNA, the polynucleotide encoding the binding site for that miRNA (ormiRNAs) would be more highly expressed. Hence, the target polypeptideencoded by the polynucleotide is selected as a protein which triggers orinduces cell death. Neighboring noncancer cells, harboring a higherexpression of the same miRNA would be less affected by the encoded deathsignal as the polynucleotide would be expressed at a lower level due tothe effects of the miRNA binding to the binding site or “sensor” encodedin the 3′-UTR. Conversely, cell survival or cytoprotective signals maybe delivered to tissues containing cancer and non-cancerous cells wherea miRNA has a higher expression in the cancer cells—the result being alower survival signal to the cancer cell and a larger survival signatureto the normal cell. Multiple polynucleotides may be designed andadministered having different signals according to the previousparadigm.

In one embodiment, the expression of a nucleic acid may be controlled byincorporating at least one sensor sequence in the nucleic acid andformulating the nucleic acid. As a non-limiting example, a nucleic acidmay be targeted to an orthotopic tumor by having a nucleic acidincorporating a miR-122 binding site and formulated in a lipidnanoparticle comprising a cationic lipid such as DLin-MC3-DMA.

According to the present invention, the polynucleotides may be alteredas to avoid the deficiencies of other polypeptide-encoding molecules ofthe art. Hence, in this embodiment the polynucleotides are referred toas alternative polynucleotides.

Through an understanding of the expression patterns of microRNA indifferent cell types, alternative nucleic acids, enhanced alternativeRNA or ribonucleic acids such as polynucleotides can be engineered formore targeted expression in specific cell types or only under specificbiological conditions. Through introduction of tissue-specific microRNAbinding sites, alternative nucleic acids, enhanced alternative RNA orribonucleic acids, could be designed that would be optimal for proteinexpression in a tissue or in the context of a biological condition.

Transfection experiments can be conducted in relevant cell lines, usingengineered alternative nucleic acids, enhanced alternative RNA orribonucleic acids and protein production can be assayed at various timepoints post-transfection. For example, cells can be transfected withdifferent microRNA binding site-engineering nucleic acids or mRNA and byusing an ELISA kit to the relevant protein and assaying protein producedat 6 hr, 12 hr, 24 hr, 48 hr, 72 hr and 7 days post-transfection. Invivo experiments can also be conducted using microRNA-bindingsite-engineered molecules to examine changes in tissue-specificexpression of formulated alternative nucleic acids, enhanced alternativeRNA or ribonucleic acids.

Non-limiting examples of cell lines which may be useful in theseinvestigations include those from ATCC (Manassas, Va.) including MRC-5,A549, T84, NCI-H2126 [H2126], NCI-H1688 [H1688], WI-38, WI-38 VA-13subline 2RA, WI-26 VA4, C3A [HepG2/C3A, derivative of Hep G2 (ATCCHB-8065)], THLE-3, H69AR, NCI-H292 [H292], CFPAC-1, NTERA-2 cl.D1[NT2/D1], DMS 79, DMS 53, DMS 153, DMS 114, MSTO-211H, SW 1573 [SW-1573,SW1573], SW 1271 [SW-1271, SW1271], SHP-77, SNU-398, SNU-449, SNU-182,SNU-475, SNU-387, SNU-423, NL20, NL20-TA [NL20T-A], THLE-2, HBE135-E6E7,HCC827, HCC4006, NCI-H23 [H23], NCI-H1299, NCI-H187 [H187], NCI-H358[H-358, H358], NCI-H378 [H378], NCI-H522 [H522], NCI-H526 [H526],NCI-H727 [H727], NCI-H810 [H810], NCI-H889 [H889], NCI-H1155 [H1155],NCI-H1404 [H1404], NCI-N87 [N87], NCI-H196 [H196], NCI-H211 [H211],NCI-H220 [H220], NCI-H250 [H250], NCI-H524 [H524], NCI-H647 [H647],NCI-H650 [H650], NCI-H711 [H711], NCI-H719 [H719], NCI-H740 [H740],NCI-H748 [H748], NCI-H774 [H774], NCI-H838 [H838], NCI-H841 [H841],NCI-H847 [H847], NCI-H865 [H865], NCI-H920 [H920], NCI-H1048 [H1048],NCI-H1092 [H1092], NCI-H1105 [H1105], NCI-H1184 [H1184], NCI-H1238[H1238], NCI-H1341 [H1341], NCI-H1385 [H1385], NCI-H1417 [H1417],NCI-H1435 [H1435], NCI-H1436 [H1436], NCI-H1437 [H1437], NCI-H1522[H1522], NCI-H1563 [H1563], NCI-H1568 [H1568], NCI-H1573 [H1573],NCI-H1581 [H1581], NCI-H1618 [H1618], NCI-H1623 [H1623], NCI-H1650[H-1650, H1650], NCI-H1651 [H1651], NCI-H1666 [H-1666, H1666], NCI-H1672[H1672], NCI-H1693 [H1693], NCI-H1694 [H1694], NCI-H1703 [H1703],NCI-H1734 [H-1734, H1734], NCI-H1755 [H1755], NCI-H1755 [H1755],NCI-H1770 [H1770], NCI-H1793 [H1793], NCI-H1836 [H1836], NCI-H1838[H1838], NCI-H1869 [H1869], NCI-H1876 [H1876], NCI-H1882 [H1882],NCI-H1915 [H1915], NCI-H1930 [H1930], NCI-H1944 [H1944], NCI-H1975[H-1975, H1975], NCI-H1993 [H1993], NCI-H2023 [H2023], NCI-H2029[H2029], NCI-H2030 [H2030], NCI-H2066 [H2066], NCI-H2073 [H2073],NCI-H2081 [H2081], NCI-H2085 [H2085], NCI-H2087 [H2087], NCI-H2106[H2106], NCI-H2110 [H2110], NCI-H2135 [H2135], NCI-H2141 [H2141],NCI-H2171 [H2171], NCI-H2172 [H2172], NCI-H2195 [H2195], NCI-H2196[H2196], NCI-H2198 [H2198], NCI-H2227 [H2227], NCI-H2228 [H2228],NCI-H2286 [H2286], NCI-H2291 [H2291], NCI-H2330 [H2330], NCI-H2342[H2342], NCI-H2347 [H2347], NCI-H2405 [H2405], NCI-H2444 [H2444],UMC-11, NCI-H64 [H64], NCI-H735 [H735], NCI-H735 [H735], NCI-H1963[H1963], NCI-H2107 [H2107], NCI-H2108 [H2108], NCI-H2122 [H2122], Hs573.T, Hs 573.Lu, PLC/PRF/5, BEAS-2B, Hep G2, Tera-1, Tera-2, NCI-H69[H69], NCI-H128 [H128], ChaGo-K-1, NCI-H446 [H446], NCI-H209 [H209],NCI-H146 [H146], NCI-H441 [H441], NCI-H82 [H82], NCI-H460 [H460],NCI-H596 [H596], NCI-H676B [H67613], NCI-H345 [H345], NCI-H820 [H820],NCI-H520 [H520], NCI-H661 [H661], NCI-H510A [H510A, NCI-H510], SK-HEP-1,A-427, Calu-1, Calu-3, Calu-6, SK-LU-1, SK-MES-1, SW 900 [SW-900,SW900], Malme-3M, and Capan-1.

In some embodiments, alternative messenger RNA can be designed toincorporate microRNA binding region sites that either have 100% identityto known seed sequences or have less than 100% identity to seedsequences. The seed sequence can be partially mutated to decreasemicroRNA binding affinity and as such result in reduced downmodulationof that mRNA transcript. In essence, the degree of match or mis-matchbetween the target mRNA and the microRNA seed can act as a rheostat tomore finely tune the ability of the microRNA to modulate proteinexpression. In addition, mutation in the non-seed region of a microRNAbinding site may also impact the ability of a microRNA to modulateprotein expression.

In one embodiment, a miR sequence may be incorporated into the loop of astem loop.

In another embodiment, a miR seed sequence may be incorporated in theloop of a stem loop and a miR binding site may be incorporated into the5′ or 3′ stem of the stem loop.

In one embodiment, a TEE may be incorporated on the 5′end of the stem ofa stem loop and a miR seed may be incorporated into the stem of the stemloop. In another embodiment, a TEE may be incorporated on the 5′end ofthe stem of a stem loop, a miR seed may be incorporated into the stem ofthe stem loop and a miR binding site may be incorporated into the 3′endof the stem or the sequence after the stem loop. The miR seed and themiR binding site may be for the same and/or different miR sequences.

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′-UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, incorporated herein byreference in its entirety).

In one embodiment, the incorporation of a miR sequence and/or a TEEsequence changes the shape of the stem loop region which may increaseand/or decrease translation. (see e.g, Kedde et al. A Pumilio-inducedRNA structure switch in p27-3′-UTR controls miR-221 and miR-22accessibility. Nature Cell Biology. 2010, incorporated herein byreference in its entirety).

In one embodiment, the 5′-UTR may comprise at least one microRNAsequence. The microRNA sequence may be, but is not limited to, a 19 or22 nucleotide sequence and/or a microRNA sequence without the seed.

In one embodiment the microRNA sequence in the 5′-UTR may be used tostabilize the nucleic acid and/or mRNA described herein.

In another embodiment, a microRNA sequence in the 5′-UTR may be used todecrease the accessibility of the site of translation initiation suchas, but not limited to a start codon. Matsuda et al (PLoS One. 201011(5):e15057; incorporated herein by reference in its entirety) usedantisense locked nucleic acid (LNA) oligonucleotides and exon-junctioncomplexes (EJCs) around a start codon (−4 to +37 where the A of the AUGcodons is +1) in order to decrease the accessibility to the first startcodon (AUG). Matsuda showed that altering the sequence around the startcodon with an LNA or EJC the efficiency, length and structural stabilityof the nucleic acid or mRNA is affected. The nucleic acids or mRNA ofthe present invention may comprise a microRNA sequence, instead of theLNA or EJC sequence described by Matsuda et al, near the site oftranslation initiation in order to decrease the accessibility to thesite of translation initiation. The site of translation initiation maybe prior to, after or within the microRNA sequence. As a non-limitingexample, the site of translation initiation may be located within amicroRNA sequence such as a seed sequence or binding site. As anothernon-limiting example, the site of translation initiation may be locatedwithin a miR-122 sequence such as the seed sequence or the mir-122binding site.

In one embodiment, the nucleic acids or mRNA of the present inventionmay include at least one microRNA in order to dampen the antigenpresentation by antigen presenting cells. The microRNA may be thecomplete microRNA sequence, the microRNA seed sequence, the microRNAsequence without the seed or a combination thereof. As a non-limitingexample, the microRNA incorporated into the nucleic acids or mRNA of thepresent invention may be specific to the hematopoietic system. Asanother non-limiting example, the microRNA incorporated into the nucleicacids or mRNA of the present invention to dampen antigen presentation ismiR-142-3p.

In one embodiment, the nucleic acids or mRNA of the present inventionmay include at least one microRNA in order to dampen expression of theencoded polypeptide in a cell of interest. As a non-limiting example,the nucleic acids or mRNA of the present invention may include at leastone miR-122 binding site in order to dampen expression of an encodedpolypeptide of interest in the liver. As another non-limiting example,the nucleic acids or mRNA of the present invention may include at leastone miR-142-3p binding site, miR-142-3p seed sequence, miR-142-3pbinding site without the seed, miR-142-5p binding site, miR-142-5p seedsequence, miR-142-5p binding site without the seed, miR-146 bindingsite, miR-146 seed sequence and/or miR-146 binding site without the seedsequence.

In one embodiment, the nucleic acids or mRNA of the present inventionmay comprise at least one microRNA binding site in the 3′-UTR in orderto selectively degrade mRNA therapeutics in the immune cells to subdueunwanted immunogenic reactions caused by therapeutic delivery. As anon-limiting example, the microRNA binding site may be the alternativenucleic acids more unstable in antigen presenting cells. Non-limitingexamples of these microRNA include mir-142-5p, mir-142-3p, mir-146a-5pand mir-146-3p.

In one embodiment, the nucleic acids or mRNA of the present inventioncomprises at least one microRNA sequence in a region of the nucleic acidor mRNA which may interact with a RNA binding protein.

RNA Motifs for RNA Binding Proteins (RBPs)

RNA binding proteins (RBPs) can regulate numerous aspects of co- andpost-transcription gene expression such as, but not limited to, RNAsplicing, localization, translation, turnover, polyadenylation, capping,alteration, export and localization. RNA-binding domains (RBDs), suchas, but not limited to, RNA recognition motif (RR) and hnRNP K-homology(KH) domains, typically regulate the sequence association between RBPsand their RNA targets (Ray et al. Nature 2013. 499:172-177; incorporatedherein by reference in its entirety). In one embodiment, the canonicalRBDs can bind short RNA sequences. In another embodiment, the canonicalRBDs can recognize structure RNAs.

In one embodiment, to increase the stability of the mRNA of interest, anmRNA encoding HuR can be co-transfected or co-injected along with themRNA of interest into the cells or into the tissue. These proteins canalso be tethered to the mRNA of interest in vitro and then administeredto the cells together. Poly A tail binding protein, PABP interacts witheukaryotic translation initiation factor eIF4G to stimulatetranslational initiation. Co-administration of mRNAs encoding these RBPsalong with the mRNA drug and/or tethering these proteins to the mRNAdrug in vitro and administering the protein-bound mRNA into the cellscan increase the translational efficiency of the mRNA. The same conceptcan be extended to co-administration of mRNA along with mRNAs encodingvarious translation factors and facilitators as well as with theproteins themselves to influence RNA stability and/or translationalefficiency.

In one embodiment, the nucleic acids and/or mRNA may comprise at leastone RNA-binding motif such as, but not limited to a RNA-binding domain(RBD).

In one embodiment, the RBD may be any of the RBDs, fragments or variantsthereof descried by Ray et al. (Nature 2013. 499:172-177; incorporatedherein by reference in its entirety).

In one embodiment, the nucleic acids or mRNA of the present inventionmay comprise a sequence for at least one RNA-binding domain (RBDs). Whenthe nucleic acids or mRNA of the present invention comprise more thanone RBD, the RBDs do not need to be from the same species or even thesame structural class.

In one embodiment, at least one flanking region (e.g., the 5′-UTR and/orthe 3′-UTR) may comprise at least one RBD. In another embodiment, thefirst flanking region and the second flanking region may both compriseat least one RBD. The RBD may be the same or each of the RBDs may haveat least 60% sequence identity to the other RBD. As a non-limitingexample, at least on RBD may be located before, after and/or within the3′-UTR of the nucleic acid or mRNA of the present invention. As anothernon-limiting example, at least one RBD may be located before or withinthe first 300 nucleosides of the 3′-UTR.

In another embodiment, the nucleic acids and/or mRNA of the presentinvention may comprise at least one RBD in the first region of linkednucleosides. The RBD may be located before, after or within a codingregion (e.g., the ORF).

In yet another embodiment, the first region of linked nucleosides and/orat least one flanking region may comprise at least on RBD. As anon-limiting example, the first region of linked nucleosides maycomprise a RBD related to splicing factors and at least one flankingregion may comprise a RBD for stability and/or translation factors.

In one embodiment, the nucleic acids and/or mRNA of the presentinvention may comprise at least one RBD located in a coding and/ornon-coding region of the nucleic acids and/or mRNA.

In one embodiment, at least one RBD may be incorporated into at leastone flanking region to increase the stability of the nucleic acid and/ormRNA of the present invention.

In one embodiment, a microRNA sequence in a RNA binding protein motifmay be used to decrease the accessibility of the site of translationinitiation such as, but not limited to a start codon. The nucleic acidsor mRNA of the present invention may comprise a microRNA sequence,instead of the LNA or EJC sequence described by Matsuda et al, near thesite of translation initiation in order to decrease the accessibility tothe site of translation initiation. The site of translation initiationmay be prior to, after or within the microRNA sequence. As anon-limiting example, the site of translation initiation may be locatedwithin a microRNA sequence such as a seed sequence or binding site. Asanother non-limiting example, the site of translation initiation may belocated within a miR-122 sequence such as the seed sequence or themir-122 binding site.

In another embodiment, an antisense locked nucleic acid (LNA)oligonucleotides and exon-junction complexes (EJCs) may be used in theRNA binding protein motif. The LNA and EJCs may be used around a startcodon (−4 to +37 where the A of the AUG codons is +1) in order todecrease the accessibility to the first start codon (AUG).

Codon Optimization

The polynucleotides of the invention, their regions or parts orsubregions may be codon optimized. Codon optimization methods are knownin the art and may be useful in efforts to achieve one or more ofseveral goals. These goals include to match codon frequencies in targetand host organisms to ensure proper folding, bias GC content to increasemRNA stability or reduce secondary structures, minimize tandem repeatcodons or base runs that may impair gene construction or expression,customize transcriptional and translational control regions, insert orremove protein trafficking sequences, remove/add post translationmodification sites in encoded protein (e.g., glycosylation sites), add,remove or shuffle protein domains, insert or delete restriction sites,modify ribosome binding sites and mRNA degradation sites, to adjusttranslational rates to allow the various domains of the protein to foldproperly, or to reduce or eliminate problem secondary structures withinthe polynucleotide. Codon optimization tools, algorithms and servicesare known in the art, non-limiting examples include services fromGeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and/orproprietary methods. In one embodiment, the ORF sequence is optimizedusing optimization algorithms. Codon options for each amino acid aregiven in Table 9.

TABLE 9 Codon Options. Single Letter Amino Acid Code Codon OptionsIsoleucine I AUU, AUC, AUA Leucine L CUU, CUC, CUA, CUG, UUA, UUG ValineV GUU, GUC, GUA, GUG Phenylalanine F UUU, UUC Methionine M AUG CysteineC UGU, UGC Alanine A GCU, GCC, GCA, GCG Glycine G GGU, GGC, GGA, GGGProline P CCU, CCC, CCA, CCG Threonine T ACU, ACC, ACA, ACG Serine SUCU, UCC, UCA, UCG, AGU, AGC Tyrosine Y UAU, UAC Tryptophan W UGGGlutamine Q CAA, CAG Asparagine N AAU, AAC Histidine H CAU, CAC Glutamicacid E GAA, GAG Aspartic acid D GAU, GAC Lysine K AAA, AAG Arginine RCGU, CGC, CGA, CGG, AGA, AGG Selenocysteine Sec UGA in mRNA in presenceof Selenocystein insertion element (SECIS) Stop codons Stop UAA, UAG,UGA

“Codon optimized” refers to the modification of a starting nucleotidesequence by replacing at least one codon of the starting nucleotidesequence with another codon encoding the same amino acid (e.g., toincrease in vivo expression). Table 10 contains the codon usagefrequency for humans (Codon usage database:[[www.]]kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=9606&aa=1&style=N).

TABLE 10 Codon usage frequency table for humans. Amino Amino Amino AminoCodon Acid % Codon Acid % Codon Acid % Codon Acid % UUU F (2) 46 UCU S(3) 19 UAU Y (2) 44 UGU C (2) 46 UUC F (1) 54 UCC S (2) 22 UAC Y (1) 56UGC C (1) 54 UUA L (5) 8 UCA S (4) 15 UAA * 30 UGA * 47 UUG L (4) 13 UCGS (6) 5 UAG * 24 UGG W (1) 100 CUU L (3) 13 CCU P (2) 29 CAU H (2) 42CGU R (6) 8 CUC L(2) 20 CCC P (1) 32 CAC H (1) 58 CGC R (4) 18 CUA L (6)7 CCA P (3) 28 CAA Q (2) 27 CGA R (5) 11 CUG L (1) 40 CCG P (4) 11 CAG Q(1) 73 CGG R (3) 20 AUU I (2) 36 ACU T (3) 25 AAU N (2) 47 AGU S (5) 15AUC I (1) 47 ACC T (1) 36 AAC N (1) 53 AGC S (1) 24 AUA I (3) 17 ACA T(2) 28 AAA K (2) 43 AGA R (2) 21 AUG M (1) 100 ACG T (4) 11 AAG K (1) 57AGG R (1) 21 GUU V (3) 18 GCU A (2) 27 GAU D (2) 46 GGU G (4) 16 GUC V(2) 24 GCC A (1) 40 GAC D (1) 54 GGC G (1) 34 GUA V (4) 12 GCA A (3) 23GAA E (2) 42 GGA G (2) 25 GUG V (1) 46 GCG A (4) 11 GAG E (1) 58 GGG G(3) 25In Table 10, the number in parentheses after the one letter amino acidcode indicates the frequency of that codon relative to other codonsencoding the same amino acid, where “1” is the highest frequency andhigher integers indicate less frequent codons.

A guanine maximized codon is a codon having the highest number ofguanines possible for a specified amino acid. A cytosine maximized codonis a codon having the highest number of cytosines possible for aspecified amino acid. A guanine/cytosine maximized codon refers to acodon having the highest number of guanines, cytosines, or combinationof guanines and cytosines for a specified amino acid. When two or morecodons have the same number of guanines, cytosines, or combinationthereof for a specified amino acid, a low frequency maximized codon is acodon that is not the highest frequency codon.

In one embodiment, after a nucleotide sequence has been codon optimizedit may be further evaluated for regions containing restriction sites. Atleast one nucleotide within the restriction site regions may be replacedwith another nucleotide in order to remove the restriction site from thesequence, but the replacement of nucleotides does not alter the aminoacid sequence which is encoded by the codon optimized nucleotidesequence.

Features, which may be considered beneficial in some embodiments of thepresent invention, may be encoded by regions of the polynucleotide andsuch regions may be upstream (5′) or downstream (3′) to a region whichencodes a polypeptide. These regions may be incorporated into thepolynucleotide before and/or after codon optimization of the proteinencoding region or open reading frame (ORF). It is not required that apolynucleotide contain both a 5′ and 3′ flanking region. Examples ofsuch features include, but are not limited to, untranslated regions(UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags andmay include multiple cloning sites which may have XbaI recognition.

In some embodiments, a 5′ UTR and/or a 3′ UTR region may be provided asflanking regions. Multiple 5′ or 3′ UTRs may be included in the flankingregions and may be the same or of different sequences. Any portion ofthe flanking regions, including none, may be codon optimized and any mayindependently contain one or more different structural or chemicalalterations, before and/or after codon optimization.

After optimization (if desired), the polynucleotides components arereconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized polynucleotide may be reconstituted and transformed intochemically competent E. coli, yeast, neurospora, maize, drosophila, etc.where high copy plasmid-like or chromosome structures occur by methodsdescribed herein.

Uses of Alternative Nucleic Acids Therapeutic Agents

The alternative nucleic acids described herein can be used astherapeutic agents. For example, an alternative nucleic acid describedherein can be administered to an animal or subject, wherein thealternative nucleic acid is translated in vivo to produce a therapeuticpeptide in the animal or subject. Accordingly, provided herein are mRNA,compositions (such as pharmaceutical compositions), methods, kits, andreagents for treatment or prevention of disease or conditions in humansand other mammals. The active therapeutic agents of the presentdisclosure include alternative nucleic acids, cells containingalternative nucleic acids or polypeptides translated from thealternative nucleic acids, polypeptides translated from alternativenucleic acids, cells contacted with cells containing alternative nucleicacids or polypeptides translated from the alternative nucleic acids,tissues containing cells containing alternative nucleic acids and organscontaining tissues containing cells containing alternative nucleicacids.

Provided are methods of inducing translation of a synthetic orrecombinant polynucleotide to produce a polypeptide in a cell populationusing the alternative nucleic acids described herein. Such translationcan be in vivo, ex vivo, in culture, or in vitro. The cell population iscontacted with an effective amount of a composition containing a nucleicacid that has at least one nucleoside alteration, and a translatableregion encoding the polypeptide. The population is contacted underconditions such that the nucleic acid is localized into one or morecells of the cell population and the recombinant polypeptide istranslated in the cell from the nucleic acid.

An effective amount of the composition is provided based, at least inpart, on the target tissue, target cell type, means of administration,physical characteristics of the nucleic acid (e.g., size, and extent ofalternative nucleosides), and other determinants. In general, aneffective amount of the composition provides efficient proteinproduction in the cell, preferably more efficient than a compositioncontaining a corresponding unaltered nucleic acid. Increased efficiencymay be demonstrated by increased cell transfection (i.e., the percentageof cells transfected with the nucleic acid), increased proteintranslation from the nucleic acid, decreased nucleic acid degradation(as demonstrated, e.g., by increased duration of protein translationfrom a modified nucleic acid), or reduced innate immune response of thehost cell or improve therapeutic utility.

Aspects of the present disclosure are directed to methods of inducing invivo translation of a recombinant polypeptide in a mammalian subject inneed thereof. Therein, an effective amount of a composition containing anucleic acid that has at least one nucleoside alteration and atranslatable region encoding the polypeptide is administered to thesubject using the delivery methods described herein. The nucleic acid isprovided in an amount and under other conditions such that the nucleicacid is localized into a cell or cells of the subject and therecombinant polypeptide is translated in the cell from the nucleic acid.The cell in which the nucleic acid is localized, or the tissue in whichthe cell is present, may be targeted with one or more than one rounds ofnucleic acid administration.

Other aspects of the present disclosure relate to transplantation ofcells containing alternative nucleic acids to a mammalian subject.Administration of cells to mammalian subjects is known to those ofordinary skill in the art, such as local implantation (e.g., topical orsubcutaneous administration), organ delivery or systemic injection(e.g., intravenous injection or inhalation), as is the formulation ofcells in pharmaceutically acceptable carrier. Compositions containingalternative nucleic acids are formulated for administrationintramuscularly, transarterially, intraperitoneally, intravenously,intranasally, subcutaneously, endoscopically, transdermally, orintrathecally. In some embodiments, the composition is formulated forextended release.

The subject to whom the therapeutic agent is administered suffers fromor is at risk of developing a disease, disorder, or deleteriouscondition. Provided are methods of identifying, diagnosing, andclassifying subjects on these bases, which may include clinicaldiagnosis, biomarker levels, genome-wide association studies (GWAS), andother methods known in the art.

In certain embodiments, the administered alternative nucleic aciddirects production of one or more recombinant polypeptides that providea functional activity which is substantially absent in the cell in whichthe recombinant polypeptide is translated. For example, the missingfunctional activity may be enzymatic, structural, or gene regulatory innature.

In other embodiments, the administered alternative nucleic acid directsproduction of one or more recombinant polypeptides that replace apolypeptide (or multiple polypeptides) that is substantially absent inthe cell in which the recombinant polypeptide is translated. Suchabsence may be due to genetic mutation of the encoding gene orregulatory pathway thereof. In other embodiments, the administeredalternative nucleic acid directs production of one or more recombinantpolypeptides to supplement the amount of polypeptide (or multiplepolypeptides) that is present in the cell in which the recombinantpolypeptide is translated. Alternatively, the recombinant polypeptidefunctions to antagonize the activity of an endogenous protein presentin, on the surface of, or secreted from the cell. Usually, the activityof the endogenous protein is deleterious to the subject, for example,due to mutation of the endogenous protein resulting in altered activityor localization. Additionally, the recombinant polypeptide antagonizes,directly or indirectly, the activity of a biological moiety present in,on the surface of, or secreted from the cell. Examples of antagonizedbiological moieties include lipids (e.g., cholesterol), a lipoprotein(e.g., low density lipoprotein), a nucleic acid, a carbohydrate, or asmall molecule toxin.

The recombinant proteins described herein are engineered forlocalization within the cell, potentially within a specific compartmentsuch as the nucleus, or are engineered for secretion from the cell ortranslocation to the plasma membrane of the cell.

As described herein, a useful feature of the alternative nucleic acidsof the present disclosure is the capacity to reduce, evade, avoid oreliminate the innate immune response of a cell to an exogenous nucleicacid. Provided are methods for performing the titration, reduction orelimination of the immune response in a cell or a population of cells.In some embodiments, the cell is contacted with a first composition thatcontains a first dose of a first exogenous nucleic acid including atranslatable region and at least one nucleoside alteration, and thelevel of the innate immune response of the cell to the first exogenousnucleic acid is determined. Subsequently, the cell is contacted with asecond composition, which includes a second dose of the first exogenousnucleic acid, the second dose containing a lesser amount of the firstexogenous nucleic acid as compared to the first dose. Alternatively, thecell is contacted with a first dose of a second exogenous nucleic acid.The second exogenous nucleic acid may contain one or more alternativenucleosides, which may be the same or different from the first exogenousnucleic acid or, alternatively, the second exogenous nucleic acid maynot contain alternative nucleosides. The steps of contacting the cellwith the first composition and/or the second composition may be repeatedone or more times. Additionally, efficiency of protein production (e.g.,protein translation) in the cell is optionally determined, and the cellmay be re-transfected with the first and/or second compositionrepeatedly until a target protein production efficiency is achieved.

Therapeutics for Diseases and Conditions

Provided are methods for treating or preventing a symptom of diseasescharacterized by missing or aberrant protein activity, by replacing themissing protein activity or overcoming the aberrant protein activity.Because of the rapid initiation of protein production followingintroduction of alternative mRNAs, as compared to viral DNA vectors, thecompounds of the present disclosure are particularly advantageous intreating acute diseases such as sepsis, stroke, and myocardialinfarction. Moreover, the lack of transcriptional regulation of thealternative mRNAs of the present disclosure is advantageous in thataccurate titration of protein production is achievable. Multiplediseases are characterized by missing (or substantially diminished suchthat proper protein function does not occur) protein activity. Suchproteins may not be present, are present in very low quantities or areessentially non-functional. The present disclosure provides a method fortreating such conditions or diseases in a subject by introducing nucleicacid or cell-based therapeutics containing the alternative nucleic acidsprovided herein, wherein the alternative nucleic acids encode for aprotein that replaces the protein activity missing from the target cellsof the subject.

Diseases characterized by dysfunctional or aberrant protein activityinclude, but not limited to, cancer and proliferative diseases, geneticdiseases (e.g., cystic fibrosis), autoimmune diseases, diabetes,neurodegenerative diseases, cardiovascular diseases, and metabolicdiseases. The present disclosure provides a method for treating suchconditions or diseases in a subject by introducing nucleic acid orcell-based therapeutics containing the alternative nucleic acidsprovided herein, wherein the alternative nucleic acids encode for aprotein that antagonizes or otherwise overcomes the aberrant proteinactivity present in the cell of the subject.

Specific examples of a dysfunctional protein are the missense ornonsense mutation variants of the cystic fibrosis transmembraneconductance regulator (CFTR) gene, which produce a dysfunctional ornonfunctional, respectively, protein variant of CFTR protein, whichcauses cystic fibrosis.

Thus, provided are methods of treating cystic fibrosis in a mammaliansubject by contacting a cell of the subject with an alternative nucleicacid having a translatable region that encodes a functional CFTRpolypeptide, under conditions such that an effective amount of the CTFRpolypeptide is present in the cell. Preferred target cells areepithelial cells, such as the lung, and methods of administration aredetermined in view of the target tissue; i.e., for lung delivery, theRNA molecules are formulated for administration by inhalation.Therefore, in certain embodiments, the polypeptide of interest encodedby the mRNA of the invention is the CTFR polypeptide and the mRNA orpharmaceutical composition of the invention is for use in treatingcystic fibrosis.

In another embodiment, the present disclosure provides a method fortreating hyperlipidemia in a subject, by introducing into a cellpopulation of the subject with an alternative mRNA molecule encodingSortilin, a protein recently characterized by genomic studies, therebyameliorating the hyperlipidemia in a subject. The SORT1 gene encodes atrans-Golgi network (TGN) transmembrane protein called Sortilin. Geneticstudies have shown that one of five individuals has a single nucleotidepolymorphism, rs12740374, in the 1p13 locus of the SORT1 gene thatpredisposes them to having low levels of low-density lipoprotein (LDL)and very-low-density lipoprotein (VLDL). Each copy of the minor allele,present in about 30% of people, alters LDL cholesterol by 8 mg/dL, whiletwo copies of the minor allele, present in about 5% of the population,lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have alsobeen shown to have a 40% decreased risk of myocardial infarction.Functional in vivo studies in mice describes that overexpression ofSORT1 in mouse liver tissue led to significantly lower LDL-cholesterollevels, as much as 80% lower, and that silencing SORT1 increased LDLcholesterol approximately 200% (Musunuru K et al. From noncoding variantto phenotype via SORT1 at the 1p13 cholesterol locus. Nature 2010; 466:714-721). Therefore, in certain embodiments, the polypeptide of interestencoded by the mRNA of the invention is Sortilin and the mRNA orpharmaceutical composition of the invention is for use in treatinghyperlipidemia.

In certain embodiments, the polypeptide of interest encoded by the mRNAof the invention is granulocyte colony-stimulating factor (GCSF), andthe mRNA or pharmaceutical composition of the invention is for use intreating a neurological disease such as cerebral ischemia, or treatingneutropenia, or for use in increasing the number of hematopoietic stemcells in the blood (e.g. before collection by leukapheresis for use inhematopoietic stem cell transplantation).

In certain embodiments, the polypeptide of interest encoded by the mRNAof the invention is erythropoietin (EPO), and the mRNA or pharmaceuticalcomposition of the invention is for use in treating anemia, inflammatorybowel disease (such as Crohn's disease and/or ulcer colitis) ormyelodysplasia.

Methods of Cellular Nucleic Acid Delivery

Methods of the present disclosure enhance nucleic acid delivery into acell population, in vivo, ex vivo, or in culture. For example, a cellculture containing a plurality of host cells (e.g., eukaryotic cellssuch as yeast or mammalian cells) is contacted with a composition thatcontains an enhanced nucleic acid having at least one nucleosidealteration and, optionally, a translatable region. The composition alsogenerally contains a transfection reagent or other compound thatincreases the efficiency of enhanced nucleic acid uptake into the hostcells. The enhanced nucleic acid exhibits enhanced retention in the cellpopulation, relative to a corresponding unaltered nucleic acid. Theretention of the enhanced nucleic acid is greater than the retention ofthe unaltered nucleic acid. In some embodiments, it is at least about50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greater than theretention of the unaltered nucleic acid. Such retention advantage may beachieved by one round of transfection with the enhanced nucleic acid, ormay be obtained following repeated rounds of transfection.

In some embodiments, the enhanced nucleic acid is delivered to a targetcell population with one or more additional nucleic acids. Such deliverymay be at the same time, or the enhanced nucleic acid is delivered priorto delivery of the one or more additional nucleic acids. The additionalone or more nucleic acids may be alternative nucleic acids or unalterednucleic acids. It is understood that the initial presence of theenhanced nucleic acids does not substantially induce an innate immuneresponse of the cell population and, moreover, that the innate immuneresponse will not be activated by the later presence of the unalterednucleic acids. In this regard, the enhanced nucleic acid may not itselfcontain a translatable region, if the protein desired to be present inthe target cell population is translated from the unaltered nucleicacids.

Targeting Moieties

In embodiments of the present disclosure, alternative nucleic acids areprovided to express a protein-binding partner or a receptor on thesurface of the cell, which functions to target the cell to a specifictissue space or to interact with a specific moiety, either in vivo or invitro. Suitable protein-binding partners include antibodies andfunctional fragments thereof, scaffold proteins, or peptides.Additionally, alternative nucleic acids can be employed to direct thesynthesis and extracellular localization of lipids, carbohydrates, orother biological moieties.

Permanent Gene Expression Silencing

A method for epigenetically silencing gene expression in a mammaliansubject, comprising a nucleic acid where the translatable region encodesa polypeptide or polypeptides capable of directing sequence-specifichistone H3 methylation to initiate heterochromatin formation and reducegene transcription around specific genes for the purpose of silencingthe gene. For example, a gain-of-function mutation in the Janus Kinase 2gene is responsible for the family of Myeloproliferative Diseases.

Pharmaceutical Compositions

Pharmaceutical compositions may optionally comprise one or moreadditional therapeutically active substances. In accordance with someembodiments, a method of administering pharmaceutical compositionscomprising an alternative nucleic acid encoding one or more proteins tobe delivered to a subject in need thereof is provided. In someembodiments, compositions are administered to humans

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions is contemplated include, but are not limited to, humansand/or other primates; mammals, including commercially relevant mammalssuch as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats;and/or birds, including commercially relevant birds such as chickens,ducks, geese, and/or turkeys.

Formulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with an excipient and/orone or more other accessory ingredients, and then, if necessary and/ordesirable, shaping and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition in accordance with the present disclosuremay be prepared, packaged, and/or sold in bulk, as a single unit dose,and/or as a plurality of single unit doses. As used herein, a “unitdose” is discrete amount of the pharmaceutical composition comprising apredetermined amount of the active ingredient. The amount of the activeingredient is generally equal to the dosage of the active ingredientwhich would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosurewill vary, depending upon the identity, size, and/or condition of thesubject treated and further depending upon the route by which thecomposition is to be administered. By way of example, the compositionmay comprise between 0.1% and 100% (w/w) active ingredient.

Pharmaceutical formulations may additionally comprise a pharmaceuticallyacceptable excipient, which, as used herein, includes any and allsolvents, dispersion media, diluents, or other liquid vehicles,dispersion or suspension aids, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's The Science and Practice of Pharmacy, 21^(st)Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md.,2006; incorporated herein by reference) discloses various excipientsused in formulating pharmaceutical compositions and known techniques forthe preparation thereof. Except insofar as any conventional excipientmedium is incompatible with a substance or its derivatives, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thispresent disclosure.

In some embodiments, a pharmaceutically acceptable excipient is at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%pure. In some embodiments, an excipient is approved for use in humansand for veterinary use. In some embodiments, an excipient is approved byUnited States Food and Drug Administration. In some embodiments, anexcipient is pharmaceutical grade. In some embodiments, an excipientmeets the standards of the United States Pharmacopoeia (USP), theEuropean Pharmacopoeia (EP), the British Pharmacopoeia, and/or theInternational Pharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutical compositions include, but are not limited to, inertdiluents, dispersing and/or granulating agents, surface active agentsand/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. Suchexcipients may optionally be included in pharmaceutical formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and/or perfuming agents can bepresent in the composition, according to the judgment of the formulator.

Exemplary diluents include, but are not limited to, calcium carbonate,sodium carbonate, calcium phosphate, dicalcium phosphate, calciumsulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose,cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc.,and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum® [magnesiumaluminum silicate]), long chain amino acid derivatives, high molecularweight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol,triacetin monostearate, ethylene glycol distearate, glycerylmonostearate, and propylene glycol monostearate, polyvinyl alcohol),carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acidpolymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives(e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [Tween®20], polyoxyethylene sorbitan [Tween®60],polyoxyethylene sorbitan monooleate [Tween®80], sorbitan monopalmitate[Span®40], sorbitan monostearate [Span®60], sorbitan tristearate[Span®65], glyceryl monooleate, sorbitan monooleate [Span® 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj®45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij®30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic®F 68, Poloxamer®188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, and mannitol); naturaland synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), andlarch arabogalactan); alginates; polyethylene oxide; polyethyleneglycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes;water; alcohol; etc.; and combinations thereof.

Exemplary preservatives may include, but are not limited to,antioxidants, chelating agents, antimicrobial preservatives, antifungalpreservatives, alcohol preservatives, acidic preservatives, and/or otherpreservatives. Exemplary antioxidants include, but are not limited to,alpha tocopherol, ascorbic acid, acorbyl palmitate, butylatedhydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodiumbisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplarychelating agents include ethylenediaminetetraacetic acid (EDTA), citricacid monohydrate, disodium edetate, dipotassium edetate, edetic acid,fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaricacid, and/or trisodium edetate. Exemplary antimicrobial preservativesinclude, but are not limited to, benzalkonium chloride, benzethoniumchloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride,chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethylalcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol,phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/orthimerosal. Exemplary antifungal preservatives include, but are notlimited to, butyl paraben, methyl paraben, ethyl paraben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate,potassium sorbate, sodium benzoate, sodium propionate, and/or sorbicacid. Exemplary alcohol preservatives include, but are not limited to,ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol,chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplaryacidic preservatives include, but are not limited to, vitamin A, vitaminC, vitamin E, beta-carotene, citric acid, acetic acid, dehydroaceticacid, ascorbic acid, sorbic acid, and/or phytic acid. Otherpreservatives include, but are not limited to, tocopherol, tocopherolacetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA),butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate(SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodiummetabisulfite, potassium sulfite, potassium metabisulfite, GlydantPlus®, Phenonip®, methylparaben, Germall®115, Germaben®II, Neolone™,Kathon™ and/or Euxyl®.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., and/orcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and/or combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups, and/or elixirs. Inaddition to active ingredients, liquid dosage forms may comprise inertdiluents commonly used in the art such as, for example, water or othersolvents, solubilizing agents and emulsifiers such as ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, oral compositions can includeadjuvants such as wetting agents, emulsifying and suspending agents,sweetening, flavoring, and/or perfuming agents. In certain embodimentsfor parenteral administration, compositions are mixed with solubilizingagents such as Cremophor®, alcohols, oils, modified oils, glycols,polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing agents, wetting agents, and/or suspendingagents. Sterile injectable preparations may be sterile injectablesolutions, suspensions, and/or emulsions in nontoxic parenterallyacceptable diluents and/or solvents, for example, as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution, U.S.P., and isotonic sodiumchloride solution. Sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose any bland fixed oil canbe employed including synthetic mono- or diglycerides. Fatty acids suchas oleic acid can be used in the preparation of injectables.

Injectable formulations can be sterilized, for example, by filtrationthrough a bacterial-retaining filter, and/or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of an active ingredient, it is oftendesirable to slow the absorption of the active ingredient fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the drug then dependsupon its rate of dissolution which, in turn, may depend upon crystalsize and crystalline form. Alternatively, delayed absorption of aparenterally administered drug form is accomplished by dissolving orsuspending the drug in an oil vehicle. Injectable depot forms are madeby forming microencapsule matrices of the drug in biodegradable polymerssuch as polylactide-polyglycolide. Depending upon the ratio of drug topolymer and the nature of the particular polymer employed, the rate ofdrug release can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing compositions with suitablenon-irritating excipients such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum or vaginal cavity andrelease the active ingredient.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, an activeingredient is mixed with at least one inert, pharmaceutically acceptableexcipient such as sodium citrate or dicalcium phosphate and/or fillersor extenders (e.g., starches, lactose, sucrose, glucose, mannitol, andsilicic acid), binders (e.g., carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g.,glycerol), disintegrating agents (e.g., agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate), solution retarding agents (e.g., paraffin), absorptionaccelerators (e.g., quaternary ammonium compounds), wetting agents(e.g., cetyl alcohol and glycerol monostearate), absorbents (e.g.,kaolin and bentonite clay), and lubricants (e.g., talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate), and mixtures thereof. In the case of capsules, tablets andpills, the dosage form may comprise buffering agents.

Solid compositions of a similar type may be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugar as well as high molecular weight polyethylene glycols and thelike. Solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally comprise opacifying agents and can be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes. Solid compositions of asimilar type may be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polyethylene glycols and the like.

Dosage forms for topical and/or transdermal administration of acomposition may include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants and/or patches. Generally, anactive ingredient is admixed under sterile conditions with apharmaceutically acceptable excipient and/or any needed preservativesand/or buffers as may be required. Additionally, the present disclosurecontemplates the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms may be prepared, for example, by dissolving and/ordispensing the compound in the proper medium. Alternatively oradditionally, rate may be controlled by either providing a ratecontrolling membrane and/or by dispersing the compound in a polymermatrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticalcompositions described herein include short needle devices such as thosedescribed in U.S. Pat. Nos. 4,886,499; 5,190,521; 5,328,483; 5,527,288;4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal compositionsmay be administered by devices which limit the effective penetrationlength of a needle into the skin, such as those described in PCTpublication WO 99/34850 and functional equivalents thereof. Jetinjection devices which deliver liquid compositions to the dermis via aliquid jet injector and/or via a needle which pierces the stratumcorneum and produces a jet which reaches the dermis are suitable. Jetinjection devices are described, for example, in U.S. Pat. Nos.5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes may be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for pulmonary administration via the buccal cavity.Such a formulation may comprise dry particles which comprise the activeingredient and which have a diameter in the range from about 0.5 nm toabout 7 nm or from about 1 nm to about 6 nm. Such compositions areconveniently in the form of dry powders for administration using adevice comprising a dry powder reservoir to which a stream of propellantmay be directed to disperse the powder and/or using a self propellingsolvent/powder dispensing container such as a device comprising theactive ingredient dissolved and/or suspended in a low-boiling propellantin a sealed container. Such powders comprise particles wherein at least98% of the particles by weight have a diameter greater than 0.5 nm andat least 95% of the particles by number have a diameter less than 7 nm.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nm and at least 90% of the particles by number have adiameter less than 6 nm. Dry powder compositions may include a solidfine powder diluent such as sugar and are conveniently provided in aunit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally thepropellant may constitute 50% to 99.9% (w/w) of the composition, andactive ingredient may constitute 0.1% to 20% (w/w) of the composition. Apropellant may further comprise additional ingredients such as a liquidnon-ionic and/or solid anionic surfactant and/or a solid diluent (whichmay have a particle size of the same order as particles comprising theactive ingredient).

Pharmaceutical compositions formulated for pulmonary delivery mayprovide an active ingredient in the form of droplets of a solutionand/or suspension. Such formulations may be prepared, packaged, and/orsold as aqueous and/or dilute alcoholic solutions and/or suspensions,optionally sterile, comprising active ingredient, and may convenientlybe administered using any nebulization and/or atomization device. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, a flavoring agent such as saccharinsodium, a volatile oil, a buffering agent, a surface active agent,and/or a preservative such as methylhydroxybenzoate. Droplets providedby this route of administration may have an average diameter in therange from about 0.1 nm to about 200 nm.

Formulations described herein as being useful for pulmonary delivery areuseful for intranasal delivery of a pharmaceutical composition. Anotherformulation suitable for intranasal administration is a coarse powdercomprising the active ingredient and having an average particle fromabout 0.2 μm to 500 μm. Such a formulation is administered in the mannerin which snuff is taken, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close to the nose.

Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofactive ingredient, and may comprise one or more of the additionalingredients described herein. A pharmaceutical composition may beprepared, packaged, and/or sold in a formulation suitable for buccaladministration. Such formulations may, for example, be in the form oftablets and/or lozenges made using conventional methods, and may, forexample, 0.1% to 20% (w/w) active ingredient, the balance comprising anorally dissolvable and/or degradable composition and, optionally, one ormore of the additional ingredients described herein. Alternately,formulations suitable for buccal administration may comprise a powderand/or an aerosolized and/or atomized solution and/or suspensioncomprising active ingredient. Such powdered, aerosolized, and/oraerosolized formulations, when dispersed, may have an average particleand/or droplet size in the range from about 0.1 nm to about 200 nm, andmay further comprise one or more of any additional ingredients describedherein.

A pharmaceutical composition may be prepared, packaged, and/or sold in aformulation suitable for ophthalmic administration. Such formulationsmay, for example, be in the form of eye drops including, for example, a0.1/1.0% (w/w) solution and/or suspension of the active ingredient in anaqueous or oily liquid excipient. Such drops may further comprisebuffering agents, salts, and/or one or more other of any additionalingredients described herein. Other opthalmically-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form and/or in a liposomal preparation.Ear drops and/or eye drops are contemplated as being within the scope ofthis present disclosure.

General considerations in the formulation and/or manufacture ofpharmaceutical agents may be found, for example, in Remington's TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro(Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporatedherein by reference).

Administration

The present disclosure provides methods comprising administering mRNA inaccordance with the present disclosure to a subject in need thereof.mRNA, or pharmaceutical, imaging, diagnostic, or prophylacticcompositions thereof, may be administered to a subject using any amountand any route of administration effective for preventing, treating,diagnosing, or imaging a disease, disorder, and/or condition (e.g., adisease, disorder, and/or condition relating to working memorydeficits). The exact amount required will vary from subject to subject,depending on the species, age, and general condition of the subject, theseverity of the disease, the particular composition, its mode ofadministration, its mode of activity, and the like. Compositions inaccordance with the present disclosure are typically formulated indosage unit form for ease of administration and uniformity of dosage. Itwill be understood, however, that the total daily usage of thecompositions of the present disclosure will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective, prophylactically effective, or appropriateimaging dose level for any particular patient will depend upon a varietyof factors including the disorder being treated and the severity of thedisorder; the activity of the specific compound employed; the specificcomposition employed; the age, body weight, general health, sex and dietof the patient; the time of administration, route of administration, andrate of excretion of the specific compound employed; the duration of thetreatment; drugs used in combination or coincidental with the specificcompound employed; and like factors well known in the medical arts.

mRNAs to be delivered and/or pharmaceutical, prophylactic, diagnostic,or imaging compositions thereof may be administered to animals, such asmammals (e.g., humans, domesticated animals, cats, dogs, mice, rats,etc.). In some embodiments, pharmaceutical, prophylactic, diagnostic, orimaging compositions thereof are administered to humans.

mRNAs to be delivered and/or pharmaceutical, prophylactic, diagnostic,or imaging compositions thereof in accordance with the presentdisclosure may be administered by any route. In some embodiments, mRNAsand/or pharmaceutical, prophylactic, diagnostic, or imaging compositionsthereof, are administered by one or more of a variety of routes,including oral, intravenous, intramuscular, intra-arterial,intramedullary, intrathecal, subcutaneous, intraventricular,transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical(e.g., by powders, ointments, creams, gels, lotions, and/or drops),mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;as an oral spray, nasal spray, and/or aerosol, and/or through a portalvein catheter. In some embodiments, mRNAs, and/or pharmaceutical,prophylactic, diagnostic, or imaging compositions thereof, areadministered by systemic intravenous injection. In specific embodiments,mRNAs and/or pharmaceutical, prophylactic, diagnostic, or imagingcompositions thereof may be administered intravenously and/or orally. Inspecific embodiments, mRNAs, and/or pharmaceutical, prophylactic,diagnostic, or imaging compositions thereof, may be administered in away which allows the mRNA to cross the blood-brain barrier, vascularbarrier, or other epithelial barrier.

However, the present disclosure encompasses the delivery of mRNAs,and/or pharmaceutical, prophylactic, diagnostic, or imaging compositionsthereof, by any appropriate route taking into consideration likelyadvances in the sciences of drug delivery.

In general the most appropriate route of administration will depend upona variety of factors including the nature of the mRNA associated with atleast one agent to be delivered (e.g., its stability in the environmentof the gastrointestinal tract, bloodstream, etc.), the condition of thepatient (e.g., whether the patient is able to tolerate particular routesof administration), etc. The present disclosure encompasses the deliveryof the pharmaceutical, prophylactic, diagnostic, or imaging compositionsby any appropriate route taking into consideration likely advances inthe sciences of drug delivery.

In certain embodiments, compositions in accordance with the presentdisclosure may be administered at dosage levels sufficient to deliverfrom about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg toabout 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, fromabout 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25mg/kg, of subject body weight per day, one or more times a day, toobtain the desired therapeutic, diagnostic, prophylactic, or imagingeffect. The desired dosage may be delivered three times a day, two timesa day, once a day, every other day, every third day, every week, everytwo weeks, every three weeks, or every four weeks. In certainembodiments, the desired dosage may be delivered using multipleadministrations (e.g., two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, or more administrations).

mRNAs may be used in combination with one or more other therapeutic,prophylactic, diagnostic, or imaging agents. By “in combination with,”it is not intended to imply that the agents must be administered at thesame time and/or formulated for delivery together, although thesemethods of delivery are within the scope of the present disclosure.Compositions can be administered concurrently with, prior to, orsubsequent to, one or more other desired therapeutics or medicalprocedures. In general, each agent will be administered at a dose and/oron a time schedule determined for that agent. In some embodiments, thepresent disclosure encompasses the delivery of pharmaceutical,prophylactic, diagnostic, or imaging compositions in combination withagents that improve their bioavailability, reduce and/or modify theirmetabolism, inhibit their excretion, and/or modify their distributionwithin the body.

It will further be appreciated that therapeutically, prophylactically,diagnostically, or imaging active agents utilized in combination may beadministered together in a single composition or administered separatelyin different compositions. In general, it is expected that agentsutilized in combination with be utilized at levels that do not exceedthe levels at which they are utilized individually. In some embodiments,the levels utilized in combination will be lower than those utilizedindividually.

The particular combination of therapies (therapeutics or procedures) toemploy in a combination regimen will take into account compatibility ofthe desired therapeutics and/or procedures and the desired therapeuticeffect to be achieved. It will also be appreciated that the therapiesemployed may achieve a desired effect for the same disorder (forexample, a composition useful for treating cancer in accordance with thepresent disclosure may be administered concurrently with achemotherapeutic agent), or they may achieve different effects (e.g.,control of any adverse effects).

Kits

The present disclosure provides a variety of kits for convenientlyand/or effectively carrying out methods of the present disclosure.Typically kits will comprise sufficient amounts and/or numbers ofcomponents to allow a user to perform multiple treatments of asubject(s) and/or to perform multiple experiments.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and a nucleic acid alteration, wherein the nucleic acid iscapable of evading or avoiding induction of an innate immune response ofa cell into which the first isolated nucleic acid is introduced, andpackaging and instructions.

In one aspect, the disclosure provides kits for protein production,comprising: a first isolated alternative nucleic acid comprising atranslatable region, provided in an amount effective to produce adesired amount of a protein encoded by the translatable region whenintroduced into a target cell; a second nucleic acid comprising aninhibitory nucleic acid, provided in an amount effective tosubstantially inhibit the innate immune response of the cell; andpackaging and instructions.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and a nucleoside alteration, wherein the nucleic acid exhibitsreduced degradation by a cellular nuclease, and packaging andinstructions.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and at least two different nucleoside alterations, wherein thenucleic acid exhibits reduced degradation by a cellular nuclease, andpackaging and instructions.

In one aspect, the disclosure provides kits for protein production,comprising a first isolated nucleic acid comprising a translatableregion and at least one nucleoside alteration, wherein the nucleic acidexhibits reduced degradation by a cellular nuclease; a second nucleicacid comprising an inhibitory nucleic acid; and packaging andinstructions.

In another aspect, the disclosure provides compositions for proteinproduction, comprising a first isolated nucleic acid comprising atranslatable region and a nucleoside alteration, wherein the nucleicacid exhibits reduced degradation by a cellular nuclease, and amammalian cell suitable for translation of the translatable region ofthe first nucleic acid.

Definitions

At various places in the present specification, substituents ofcompounds of the present disclosure are disclosed in groups or inranges. It is specifically intended that the present disclosure includeeach and every individual subcombination of the members of such groupsand ranges. For example, the term “C₁₋₆ alkyl” is specifically intendedto individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl,and C₆ alkyl.

Aberrant transcription product: As used herein, the term “aberranttranscription product” refers to any contaminating transcription productor impurity that differs from the intended high fidelity RNA transcriptthat is encoded by a given DNA template. Such aberrant transcriptionproducts can include short RNAs as a result of abortive transcriptioninitiation events (Milligan et al., 1987, Nucleic Acids Res15:8783-8798) and double stranded (ds)RNAs generated by RNA dependentRNA polymerase activity (Arnaud-Barbe et al, 1998, Nucleic Acids Res26:3550-3554), RNA-primed transcription from RNA templates (Nacheva andBerzal-Herranz, 2003, Eur J Biochem 270: 1458-1465), andself-complementary 3′ extension (Triana-Alonso et al., 1995, J Biol Chem270:6298-6307), i.e. a “3′-transcript extension region”.

About: As used herein, the term “about” when used in the context of theamount of an alternative nucleobase or nucleoside in a polynucleic acidmeans +/−10% of the recited value. For example, a polynucleotidecontaining about 25% of an alternative uracil includes between22.5-27.5% of the alternative uracil.

Administered in combination: As used herein, the term “administered incombination” or “combined administration” means that two or more agentsare administered to a subject at the same time or within an intervalsuch that there may be an overlap of an effect of each agent on thepatient. In some embodiments, they are administered within about 60, 30,15, 10, 5, or 1 minute of one another. In some embodiments, theadministrations of the agents are spaced sufficiently closely togethersuch that a combinatorial (e.g., a synergistic) effect is achieved.

Altered: As used herein “altered” refers to a changed state or structureof a molecule of the invention. Molecules may be altered in many waysincluding chemically, structurally, and functionally. In one embodiment,the mRNA molecules of the present invention are altered by theintroduction of non-natural nucleosides and/or nucleotides, e.g., as itrelates to the natural ribonucleotides A, U, G, and C. Noncanonicalnucleotides such as the cap structures are not considered “altered”although they differ from the chemical structure of the A, C, G, Uribonucleotides.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans at anystage of development. In some embodiments, “animal” refers to non-humananimals at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In someembodiments, animals include, but are not limited to, mammals, birds,reptiles, amphibians, fish, and worms. In some embodiments, the animalis a transgenic animal, genetically-engineered animal, or a clone.

Antigens of interest or desired antigens: As used herein, the terms“antigens of interest” or “desired antigens” include those proteins andother biomolecules provided herein that are immunospecifically bound bythe antibodies and fragments, mutants, variants, and alterations thereofdescribed herein. Examples of antigens of interest include, but are notlimited to, insulin, insulin-like growth factor, hGH, tPA, cytokines,such as interleukins (IL), e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16,IL-17, IL-18, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega orIFN tau, tumor necrosis factor (TNF), such as TNF alpha and TNF beta,TNF gamma, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest other than the amount of analternative nucleobase or nucleoside in a polynucleic acid, refers to avalue that is similar to a stated reference value. In certainembodiments, the term “approximately” or “about” refers to a range ofvalues that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in eitherdirection (greater than or less than) of the stated reference valueunless otherwise stated or otherwise evident from the context (exceptwhere such number would exceed 100% of a possible value).

Associated with: As used herein, the terms “associated with,”“conjugated,” “linked,” “attached,” and “tethered,” when used withrespect to two or more moieties, means that the moieties are physicallyassociated or connected with one another, either directly or via one ormore additional moieties that serves as a linking agent, to form astructure that is sufficiently stable so that the moieties remainphysically associated under the conditions in which the structure isused, e.g., physiological conditions. An “association” need not bestrictly through direct covalent chemical bonding. It may also suggestionic or hydrogen bonding or a hybridization based connectivitysufficiently stable such that the “associated” entities remainphysically associated.

Biocompatible: As used herein, the term “biocompatible” means compatiblewith living cells, tissues, organs or systems posing little to no riskof injury, toxicity or rejection by the immune system.

Biodegradable: As used herein, the term “biodegradable” means capable ofbeing broken down into innocuous products by the action of livingthings.

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any substance that has activity in abiological system and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments, apolynucleotide of the present invention may be considered biologicallyactive if even a portion of the polynucleotide is biologically active ormimics an activity considered biologically relevant.

Compound: As used herein, the term “compound,” is meant to include allstereoisomers, geometric isomers, tautomers, and isotopes of thestructures depicted.

The compounds described herein can be asymmetric (e.g., having one ormore stereocenters). All stereoisomers, such as enantiomers anddiastereomers, are intended unless otherwise indicated. Compounds of thepresent disclosure that contain asymmetrically substituted carbon atomscan be isolated in optically active or racemic forms. Methods on how toprepare optically active forms from optically active starting materialsare known in the art, such as by resolution of racemic mixtures or bystereoselective synthesis. Many geometric isomers of olefins, C═N doublebonds, and the like can also be present in the compounds describedherein, and all such stable isomers are contemplated in the presentdisclosure. Cis and trans geometric isomers of the compounds of thepresent disclosure are described and may be isolated as a mixture ofisomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms.Tautomeric forms result from the swapping of a single bond with anadjacent double bond and the concomitant migration of a proton.Tautomeric forms include prototropic tautomers which are isomericprotonation states having the same empirical formula and total charge.Examples prototropic tautomers include ketone-enol pairs, amide-imidicacid pairs, lactam-lactim pairs, amide-imidic acid pairs, enamine-iminepairs, and annular forms where a proton can occupy two or more positionsof a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole.Tautomeric forms can be in equilibrium or sterically locked into oneform by appropriate substitution.

Compounds of the present disclosure also include all of the isotopes ofthe atoms occurring in the intermediate or final compounds. “Isotopes”refers to atoms having the same atomic number but different mass numbersresulting from a different number of neutrons in the nuclei. Forexample, isotopes of hydrogen include tritium and deuterium.

The compounds and salts of the present disclosure can be prepared incombination with solvent or water molecules to form solvates andhydrates by routine methods.

Conserved: As used herein, the term “conserved” refers to nucleotides oramino acid residues of a polynucleotide sequence or polypeptidesequence, respectively, that are those that occur unaltered in the sameposition of two or more sequences being compared. Nucleotides or aminoacids that are relatively conserved are those that are conserved amongstmore related sequences than nucleotides or amino acids appearingelsewhere in the sequences.

In some embodiments, two or more sequences are said to be “completelyconserved” if they are 100% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are at least 70% identical, at least 80% identical, at least 90%identical, or at least 95% identical to one another. In someembodiments, two or more sequences are said to be “highly conserved” ifthey are about 70% identical, about 80% identical, about 90% identical,about 95%, about 98%, or about 99% identical to one another. In someembodiments, two or more sequences are said to be “conserved” if theyare at least 30% identical, at least 40% identical, at least 50%identical, at least 60% identical, at least 70% identical, at least 80%identical, at least 90% identical, or at least 95% identical to oneanother. In some embodiments, two or more sequences are said to be“conserved” if they are about 30% identical, about 40% identical, about50% identical, about 60% identical, about 70% identical, about 80%identical, about 90% identical, about 95% identical, about 98%identical, or about 99% identical to one another. Conservation ofsequence may apply to the entire length of an oligonucleotide orpolypeptide or may apply to a portion, region or feature thereof.

Cyclic or Cyclized: As used herein, the term “cyclic” refers to thepresence of a continuous loop. Cyclic molecules need not be circular,only joined to form an unbroken chain of subunits. Cyclic molecules suchas the mRNA of the present invention may be single units or multimers orcomprise one or more components of a complex or higher order structure.

Cytostatic: As used herein, “cytostatic” refers to inhibiting, reducing,suppressing the growth, division, or multiplication of a cell (e.g., amammalian cell (e.g., a human cell)), bacterium, virus, fungus,protozoan, parasite, prion, or a combination thereof.

Cytotoxic: As used herein, “cytotoxic” refers to killing or causinginjurious, toxic, or deadly effect on a cell (e.g., a mammalian cell(e.g., a human cell)), bacterium, virus, fungus, protozoan, parasite,prion, or a combination thereof.

Delivery: As used herein, “delivery” refers to the act or manner ofdelivering a compound, substance, entity, moiety, cargo or payload.

Delivery Agent: As used herein, “delivery agent” refers to any substancewhich facilitates, at least in part, the in vivo delivery of apolynucleotide to targeted cells.

Destabilized: As used herein, the term “destable,” “destabilize,” or“destabilizing region” means a region or molecule that is less stablethan a starting, wild-type or native form of the same region ormolecule.

Detectable label: As used herein, “detectable label” refers to one ormore markers, signals, or moieties which are attached, incorporated orassociated with another entity that is readily detected by methods knownin the art including radiography, fluorescence, chemiluminescence,enzymatic activity, absorbance and the like. Detectable labels includeradioisotopes, fluorophores, chromophores, enzymes, dyes, metal ions,ligands such as biotin, avidin, streptavidin and haptens, quantum dots,and the like. Detectable labels may be located at any position in thepeptides or proteins disclosed herein. They may be within the aminoacids, the peptides, or proteins, or located at the N- or C-termini.

Digest: As used herein, the term “digest” means to break apart intosmaller pieces or components. When referring to polypeptides orproteins, digestion results in the production of peptides.

Distal: As used herein, the term “distal” means situated away from thecenter or away from a point or region of interest.

Encoded protein cleavage signal: As used herein, “encoded proteincleavage signal” refers to the nucleotide sequence which encodes aprotein cleavage signal.

Engineered: As used herein, embodiments of the invention are“engineered” when they are designed to have a feature or property,whether structural or chemical, that varies from a starting point,wild-type or native molecule.

Expression: As used herein, “expression” of a nucleic acid sequencerefers to one or more of the following events: (1) production of an RNAtemplate from a DNA sequence (e.g., by transcription); (2) processing ofan RNA transcript (e.g., by splicing, editing, 5′ cap formation, and/or3′ end processing); (3) translation of an RNA into a polypeptide orprotein; and (4) post-translational modification of a polypeptide orprotein.

Feature: As used herein, a “feature” refers to a characteristic, aproperty, or a distinctive element.

Formulation: As used herein, a “formulation” includes at least apolynucleotide and a delivery agent.

Fragment: A “fragment,” as used herein, refers to a portion. Forexample, fragments of proteins may comprise polypeptides obtained bydigesting full-length protein isolated from cultured cells.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Homology: As used herein, the term “homology” refers to the overallrelatedness between polymeric molecules, e.g. between nucleic acidmolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). In accordance with the invention, twopolynucleotide sequences are considered to be homologous if thepolypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%,95%, or even 99% for at least one stretch of at least about 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Inaccordance with the invention, two protein sequences are considered tobe homologous if the proteins are at least about 50%, 60%, 70%, 80%, or90% identical for at least one stretch of at least about 20 amino acids.

Identity: As used herein, the term “identity” refers to the overallrelatedness between polymeric molecules, e.g., between oligonucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of the percent identity of twopolynucleotide sequences, for example, can be performed by aligning thetwo sequences for optimal comparison purposes (e.g., gaps can beintroduced in one or both of a first and a second nucleic acid sequencesfor optimal alignment and non-identical sequences can be disregarded forcomparison purposes). In certain embodiments, the length of a sequencealigned for comparison purposes is at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least95%, or 100% of the length of the reference sequence. The nucleotides atcorresponding nucleotide positions are then compared. When a position inthe first sequence is occupied by the same nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which needs to be introduced for optimal alignment of the twosequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm. For example, the percent identity between two nucleotidesequences can be determined using methods such as those described inComputational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991;each of which is incorporated herein by reference. For example, thepercent identity between two nucleotide sequences can be determinedusing the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), whichhas been incorporated into the ALIGN program (version 2.0) using aPAM120 weight residue table, a gap length penalty of 12 and a gappenalty of 4. The percent identity between two nucleotide sequences can,alternatively, be determined using the GAP program in the GCG softwarepackage using an NWSgapdna.CMP matrix. Methods commonly employed todetermine percent identity between sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM JApplied Math., 48:1073 (1988); incorporated herein by reference.Techniques for determining identity are codified in publicly availablecomputer programs. Exemplary computer software to determine homologybetween two sequences include, but are not limited to, GCG programpackage, Devereux, J., et al., Nucleic Acids Research, 12(1), 387(1984)), BLASTP, BLASTN, and FASTAn altschul, S. F. et al., J. Molec.Biol., 215, 403 (1990)).

Inhibit expression of a gene: As used herein, the phrase “inhibitexpression of a gene” means to cause a reduction in the amount of anexpression product of the gene. The expression product can be an RNAtranscribed from the gene (e.g., an mRNA) or a polypeptide translatedfrom an mRNA transcribed from the gene. Typically a reduction in thelevel of an mRNA results in a reduction in the level of a polypeptidetranslated therefrom. The level of expression may be determined usingstandard techniques for measuring mRNA or protein.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, in a Petri dish, etc., rather than within anorganism (e.g., animal, plant, or microbe).

In vitro synthesis: As used herein, the term “in vitro synthesis” refersto an extracellular method of synthesis of mRNA.

In vivo: As used herein, the term “in vivo” refers to events that occurwithin an organism (e.g., animal, plant, or microbe or cell or tissuethereof).

Isolated: As used herein, the term “isolated” refers to a substance orentity that has been separated from at least some of the components withwhich it was associated (whether in nature or in an experimentalsetting). Isolated substances may have varying levels of purity inreference to the substances from which they have been associated.Isolated substances and/or entities may be separated from at least about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90%, or more of the other components with which theywere initially associated. In some embodiments, isolated agents are morethan about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, ormore than about 99% pure. As used herein, a substance is “pure” if it issubstantially free of other components. Substantially isolated: By“substantially isolated” is meant that the compound is substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compound of the present disclosure. Substantial separation caninclude compositions containing at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 97%, or at least about 99% by weight of thecompound of the present disclosure, or salt thereof. Methods forisolating compounds and their salts are routine in the art.

Linker: As used herein, a linker refers to a group of atoms, e.g.,10-1,000 atoms, and can be comprised of the atoms or groups such as, butnot limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide,sulfonyl, carbonyl, and imine. The linker can be attached to analternative nucleoside or nucleotide on the nucleobase or sugar moietyat a first end, and to a payload, e.g., a detectable or therapeuticagent, at a second end. The linker may be of sufficient length as to notinterfere with incorporation into a nucleic acid sequence. The linkercan be used for any useful purpose, such as to form multimers (e.g.,through linkage of two or more polynucleotides) or conjugates, as wellas to administer a payload, as described herein. Examples of chemicalgroups that can be incorporated into the linker include, but are notlimited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether,ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of whichcan be optionally substituted, as described herein. Examples of linkersinclude, but are not limited to, unsaturated alkanes, polyethyleneglycols (e.g., ethylene or propylene glycol monomeric units, e.g.,diethylene glycol, dipropylene glycol, triethylene glycol, tripropyleneglycol, tetraethylene glycol, or tetraethylene glycol), and dextranpolymers, Other examples include, but are not limited to, cleavablemoieties within the linker, such as, for example, a disulfide bond(—S—S—) or an azo bond (—N═N—), which can be cleaved using a reducingagent or photolysis. Non-limiting examples of a selectively cleavablebond include an amido bond can be cleaved for example by the use oftris(2-carboxyethyl)phosphine (TCEP), or other reducing agents, and/orphotolysis, as well as an ester bond can be cleaved for example byacidic or basic hydrolysis.

Maximized codons: As used herein the term “maximized codon” refers to acodon with the highest number of a nucleotide. For example, a “guaninemaximized codon” is the codon for a particular amino acid that has thehighest number of guanines.

Naturally occurring: As used herein, “naturally occurring” meansexisting in nature without artificial aid.

Non-human vertebrate: As used herein, a “non human vertebrate” includesall vertebrates except Homo sapiens, including wild and domesticatedspecies. Examples of non-human vertebrates include, but are not limitedto, mammals, such as alpaca, banteng, bison, camel, cat, cattle, deer,dog, donkey, gayal, goat, guinea pig, horse, llama, mule, pig, rabbit,reindeer, sheep water buffalo, and yak.

Off-target: As used herein, “off target” refers to any unintended effecton any one or more target, gene, or cellular transcript.

Open reading frame: As used herein, “open reading frame” or “ORF” refersto a sequence which does not contain a stop codon in a given readingframe.

Operably linked: As used herein, the phrase “operably linked” refers toa functional connection between two or more molecules, constructs,transcripts, entities, moieties or the like.

Paratope: As used herein, a “paratope” refers to the antigen-bindingsite of an antibody.

Patient: As used herein, “patient” refers to a subject who may seek orbe in need of treatment, requires treatment, is receiving treatment,will receive treatment, or a subject who is under care by a trainedprofessional for a particular disease or condition.

Optionally substituted: Herein a phrase of the form “optionallysubstituted X” (e.g., optionally substituted alkyl) is intended to beequivalent to “X, wherein X is optionally substituted” (e.g., “alkyl,wherein said alkyl is optionally substituted”). It is not intended tomean that the feature “X” (e.g. alkyl) per se is optional.

Peptide: As used herein, “peptide” is less than or equal to 50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long.

Pharmaceutically acceptable: The phrase “pharmaceutically acceptable” isemployed herein to refer to those compounds, materials, compositions,and/or dosage forms which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem or complication, commensurate with a reasonablebenefit/risk ratio.

Pharmaceutically acceptable excipients: The phrase “pharmaceuticallyacceptable excipient,” as used herein, refers any ingredient other thanthe compounds described herein (for example, a vehicle capable ofsuspending or dissolving the active compound) and having the propertiesof being substantially nontoxic and non-inflammatory in a patient.Excipients may include, for example: antiadherents, antioxidants,binders, coatings, compression aids, disintegrants, dyes (colors),emollients, emulsifiers, fillers (diluents), film formers or coatings,flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, and waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Pharmaceutically acceptable salts: The present disclosure also includespharmaceutically acceptable salts of the compounds described herein. Asused herein, “pharmaceutically acceptable salts” refers to derivativesof the disclosed compounds wherein the parent compound is altered byconverting an existing acid or base moiety to its salt form (e.g., byreacting the free base group with a suitable organic acid). Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines; alkalior organic salts of acidic residues such as carboxylic acids; and thelike. Representative acid addition salts include acetate, adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts, and thelike. Representative alkali or alkaline earth metal salts includesodium, lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like. The pharmaceutically acceptable salts of the presentdisclosure include the conventional non-toxic salts of the parentcompound formed, for example, from non-toxic inorganic or organic acids.The pharmaceutically acceptable salts of the present disclosure can besynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts can beprepared by reacting the free acid or base forms of these compounds witha stoichiometric amount of the appropriate base or acid in water or inan organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington's TheScience and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro(Lippincott, Williams & Wilkins, Baltimore, Md., 2006); PharmaceuticalSalts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth(eds.), Wiley-VCH, 2008, and Berge et al., Journal of PharmaceuticalScience, 66, 1-19 (1977), each of which is incorporated herein byreference in its entirety.

Pharmacokinetic: As used herein, “pharmacokinetic” refers to any one ormore properties of a molecule or compound as it relates to thedetermination of the fate of substances administered to a livingorganism. Pharmacokinetics is divided into several areas including theextent and rate of absorption, distribution, metabolism and excretion.This is commonly referred to as ADME where: (A) Absorption is theprocess of a substance entering the blood circulation; (D) Distributionis the dispersion or dissemination of substances throughout the fluidsand tissues of the body; (M) Metabolism (or Biotransformation) is theirreversible transformation of parent compounds into daughtermetabolites; and (E) Excretion (or Elimination) refers to theelimination of the substances from the body. In rare cases, some drugsirreversibly accumulate in body tissue.

Pharmaceutically acceptable solvate: The term “pharmaceuticallyacceptable solvate,” as used herein, means a compound of the inventionwherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. For example, solvates may be prepared by crystallization,recrystallization, or precipitation from a solution that includesorganic solvents, water, or a mixture thereof. Examples of suitablesolvents are ethanol, water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-rdimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

Physicochemical: As used herein, “physicochemical” means of or relatingto a physical and/or chemical property.

Preventing: As used herein, the term “preventing” refers to partially orcompletely delaying onset of an infection, disease, disorder and/orcondition; partially or completely delaying onset of one or moresymptoms, features, or clinical manifestations of a particularinfection, disease, disorder, and/or condition; partially or completelydelaying onset of one or more symptoms, features, or manifestations of aparticular infection, disease, disorder, and/or condition; partially orcompletely delaying progression from an infection, a particular disease,disorder and/or condition; and/or decreasing the risk of developingpathology associated with the infection, the disease, disorder, and/orcondition.

Prodrug: The present disclosure also includes prodrugs of the compoundsdescribed herein. As used herein, “prodrugs” refer to any substance,molecule or entity which is in a form predicate for that substance,molecule or entity to act as a therapeutic upon chemical or physicalalteration. Prodrugs may by covalently bonded or sequestered in some wayand which release or are converted into the active drug moiety prior to,upon or after administered to a mammalian subject. Prodrugs can beprepared by modifying functional groups present in the compounds in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to the parent compounds. Prodrugs include compounds whereinhydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any groupthat, when administered to a mammalian subject, cleaves to form a freehydroxyl, amino, sulfhydryl, or carboxyl group respectively. Preparationand use of prodrugs is discussed in T. Higuchi and V. Stella, “Pro-drugsas Novel Delivery Systems,” Vol. 14 of the A.C.S. Symposium Series, andin Bioreversible Carriers in Drug Design, ed. Edward B. Roche, AmericanPharmaceutical Association and Pergamon Press, 1987, both of which arehereby incorporated by reference in their entirety.

Proliferate: As used herein, the term “proliferate” means to grow,expand or increase or cause to grow, expand or increase rapidly.“Proliferative” means having the ability to proliferate.“Anti-proliferative” means having properties counter to or inapposite toproliferative properties.

Protein cleavage site: As used herein, “protein cleavage site” refers toa site where controlled cleavage of the amino acid chain can beaccomplished by chemical, enzymatic or photochemical means.

Protein cleavage signal: As used herein “protein cleavage signal” refersto at least one amino acid that flags or marks a polypeptide forcleavage.

Protein of interest: As used herein, the terms “proteins of interest” or“desired proteins” include those provided herein and fragments, mutants,variants, and alterations thereof.

Proximal: As used herein, the term “proximal” means situated nearer tothe center or to a point or region of interest.

Purified: As used herein, “purify,” “purified,” “purification” means tomake substantially pure or clear from unwanted components, materialdefilement, admixture or imperfection.

Sample: As used herein, the term “sample” or “biological sample” refersto a subset of its tissues, cells or component parts (e.g. body fluids,including but not limited to blood, mucus, lymphatic fluid, synovialfluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood,urine, vaginal fluid and semen). A sample further may include ahomogenate, lysate or extract prepared from a whole organism or a subsetof its tissues, cells or component parts, or a fraction or portionthereof, including but not limited to, for example, plasma, serum,spinal fluid, lymph fluid, the external sections of the skin,respiratory, intestinal, and genitourinary tracts, tears, saliva, milk,blood cells, tumors, organs. A sample further refers to a medium, suchas a nutrient broth or gel, which may contain cellular components, suchas proteins or nucleic acid molecule.

Signal Sequences: As used herein, the phrase “signal sequences” refersto a sequence which can direct the transport or localization of aprotein.

Significant or Significantly: As used herein, the terms “significant” or“significantly” are used synonymously with the term “substantially.”

Single unit dose: As used herein, a “single unit dose” is a dose of anytherapeutic administed in one dose/at one time/single route/single pointof contact, i.e., single administration event.

Similarity: As used herein, the term “similarity” refers to the overallrelatedness between polymeric molecules, e.g. between polynucleotidemolecules (e.g. DNA molecules and/or RNA molecules) and/or betweenpolypeptide molecules. Calculation of percent similarity of polymericmolecules to one another can be performed in the same manner as acalculation of percent identity, except that calculation of percentsimilarity takes into account conservative substitutions as isunderstood in the art.

Split dose: As used herein, a “split dose” is the division of singleunit dose or total daily dose into two or more doses.

Stable: As used herein “stable” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and preferably capable of formulation into anefficacious therapeutic agent.

Stabilized: As used herein, the term “stabilize”, “stabilized,”“stabilized region” means to make or become stable.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition in accordance with the invention may beadministered, e.g., for experimental, diagnostic, prophylactic, and/ortherapeutic purposes. Typical subjects include animals (e.g., mammalssuch as mice, rats, rabbits, non-human primates, and humans) and/orplants.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially equal: As used herein as it relates to time differencesbetween doses, the term means plus/minus 2%.

Substantially simultaneously: As used herein and as it relates toplurality of doses, the term means within 2 seconds.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of a disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with and/or may notexhibit symptoms of the disease, disorder, and/or condition but harborsa propensity to develop a disease or its symptoms. In some embodiments,an individual who is susceptible to a disease, disorder, and/orcondition (for example, cancer) may be characterized by one or more ofthe following: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinand/or nucleic acid associated with the disease, disorder, and/orcondition; (4) habits and/or lifestyles associated with development ofthe disease, disorder, and/or condition; (5) a family history of thedisease, disorder, and/or condition; and (6) exposure to and/orinfection with a microbe associated with development of the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition.

Synthetic: The term “synthetic” means produced, prepared, and/ormanufactured by the hand of man. Synthesis of polynucleotides orpolypeptides or other molecules of the present invention may be chemicalor enzymatic.

Targeted Cells: As used herein, “targeted cells” refers to any one ormore cells of interest. The cells may be found in vitro, in vivo, insitu or in the tissue or organ of an organism. The organism may be ananimal, preferably a mammal, more preferably a human and most preferablya patient.

Theoretical Minimum: The term “theoretical minimum” refers to anucleotide sequence with all of the codons in the open reading framereplaced to minimize the number of uracils in the sequence.

Therapeutic Agent: The term “therapeutic agent” refers to any agentthat, when administered to a subject, has a therapeutic, diagnostic,and/or prophylactic effect and/or elicits a desired biological and/orpharmacological effect.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of an agent to bedelivered (e.g., nucleic acid, drug, therapeutic agent, diagnosticagent, prophylactic agent, etc.) that is sufficient, when administeredto a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Therapeutically effective outcome: As used herein, the term“therapeutically effective outcome” means an outcome that is sufficientin a subject suffering from or susceptible to an infection, disease,disorder, and/or condition, to treat, improve symptoms of, diagnose,prevent, and/or delay the onset of the infection, disease, disorder,and/or condition.

Total daily dose: As used herein, a “total daily dose” is an amountgiven or prescribed in 24 hr period. It may be administered as a singleunit dose.

Transcription factor: As used herein, the term “transcription factor”refers to a DNA-binding protein that regulates transcription of DNA intoRNA, for example, by activation or repression of transcription. Sometranscription factors effect regulation of transcription alone, whileothers act in concert with other proteins. Some transcription factor canboth activate and repress transcription under certain conditions. Ingeneral, transcription factors bind a specific target sequence orsequences highly similar to a specific consensus sequence in aregulatory region of a target gene. Transcription factors may regulatetranscription of a target gene alone or in a complex with othermolecules.

Treating: As used herein, the term “treating” refers to partially orcompletely alleviating, ameliorating, improving, relieving, delayingonset of, inhibiting progression of, reducing severity of, and/orreducing incidence of one or more symptoms or features of a particularinfection, disease, disorder, and/or condition. For example, “treating”cancer may refer to inhibiting survival, growth, and/or spread of atumor. Treatment may be administered to a subject who does not exhibitsigns of a disease, disorder, and/or condition and/or to a subject whoexhibits only early signs of a disease, disorder, and/or condition forthe purpose of decreasing the risk of developing pathology associatedwith the disease, disorder, and/or condition.

Unaltered: As used herein, “unaltered” refers to any substance, compoundor molecule prior to being changed in any way. Unaltered may, but doesnot always, refer to the wild-type or native form of a biomolecule.Molecules may undergo a series of alterations whereby each alternativemolecule may serve as the “unaltered” starting molecule for a subsequentalteration.

Uracil Content: As used herein, “uracil content” refers to the numberand/or distribution of uracils in a particular sequence, e.g., an openreading frame.

Wild-type Sequence: As used herein, a “wild-type sequence” is thesequence of the naturally occurring mRNA that encodes the polypeptide ofinterest.

Equivalents and Scope

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments in accordance with the invention described herein. The scopeof the present invention is not intended to be limited to the aboveDescription, but rather is as set forth in the appended claims.

In the claims, articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Claims or descriptions that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

It is also noted that the term “comprising” is intended to be open andpermits but does not require the inclusion of additional elements orsteps. When the term “comprising” is used herein, the term “consistingof” is thus also encompassed and disclosed.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the compositions of the invention (e.g., anynucleic acid or protein encoded thereby; any method of production; anymethod of use; etc.) can be excluded from any one or more claims, forany reason, whether or not related to the existence of prior art.

EXAMPLES

The present disclosure is further described in the following examples,which do not limit the scope of the disclosure described in the claims.

Example 1: PCR for cDNA Production

PCR procedures for the preparation of cDNA are performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). This systemincludes 2×KAPA ReadyMix 12.5 μl; Forward Primer (10 uM) 0.75 μl;Reverse Primer (10 uM) 0.75 μl; Template cDNA 100 ng; and dH₂O dilutedto 25.0 μl. The reaction conditions are at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention incorporates a poly-T₁₂₀ fora poly-A₁₂₀ in the mRNA. Other reverse primers with longer or shorterpoly-T tracts can be used to adjust the length of the poly-A tail in themRNA.

The reaction is cleaned up using Invitrogen's PURELINK™ PCR Micro Kit(Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA is then submitted for sequencing analysis beforeproceeding to the in vitro transcription reaction.

Example 2. In Vitro Transcription (IVT) A. Materials and Methods

Alternative mRNAs according to the invention are made using standardlaboratory methods and materials for in vitro transcription with theexception that the nucleotide mix contains alternative nucleotides. Theopen reading frame (ORF) of the gene of interest may be flanked by a 5′untranslated region (UTR) containing a strong Kozak translationalinitiation signal and an alpha-globin 3′ UTR terminating with anoligo(dT) sequence for templated addition of a polyA tail for mRNAs notincorporating adenosine analogs. Adenosine-containing mRNAs aresynthesized without an oligo (dT) sequence to allow forpost-transcription poly (A) polymerase poly-(A) tailing.

The ORF may also include various upstream or downstream additions (suchas, but not limited to, β-globin, tags, etc.) may be ordered from anoptimization service such as, but limited to, DNA2.0 (Menlo Park,Calif.) and may contain multiple cloning sites which may have XbaIrecognition. Upon receipt of the construct, it may be reconstituted andtransformed into chemically competent E. coli.

For the present invention, NEB DH5-alpha Competent E. coli may be used.Transformations are performed according to NEB instructions using 100 ngof plasmid. The protocol is as follows:

Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10minutes.

Add 1-5 μl containing 1 pg-100 ng of plasmid DNA to the cell mixture.Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

Place the mixture on ice for 30 minutes. Do not mix.

Heat shock at 42° C. for exactly 30 seconds. Do not mix.

Place on ice for 5 minutes. Do not mix.

Pipette 950 μl of room temperature SOC into the mixture.

Place at 37° C. for 60 minutes. Shake vigorously (250 rpm) or rotate.

Warm selection plates to 37° C.

Mix the cells thoroughly by flicking the tube and inverting.

Spread 50-100 μl of each dilution onto a selection plate and incubateovernight at 37° C. Alternatively, incubate at 30° C. for 24-36 hours or25° C. for 48 hours.

A single colony is then used to inoculate 5 ml of LB growth media usingthe appropriate antibiotic and then allowed to grow (250 RPM, 37° C.)for 5 hours. This is then used to inoculate a 200 ml culture medium andallowed to grow overnight under the same conditions.

To isolate the plasmid (up to 850 μg), a maxi prep is performed usingthe Invitrogen PURELINK™ HiPure Maxiprep Kit (Carlsbad, Calif.),following the manufacturer's instructions.

In order to generate cDNA for In Vitro Transcription (IVT), the plasmidis first linearized using a restriction enzyme such as XbaI. A typicalrestriction digest with XbaI will comprise the following: Plasmid 1.0μg; 10× Buffer 1.0 μl; XbaI 1.5 μl; dH₂O up to 10 μl; incubated at 37°C. for 1 hr. If performing at lab scale (<5 μg), the reaction is cleanedup using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, Calif.) permanufacturer's instructions. Larger scale purifications may need to bedone with a product that has a larger load capacity such as Invitrogen'sstandard PURELINK™ PCR Kit (Carlsbad, Calif.). Following the cleanup,the linearized vector is quantified using the NanoDrop and analyzed toconfirm linearization using agarose gel electrophoresis.

IVT Reaction

The in vitro transcription reaction generates mRNA containingalternative nucleotides or alternative RNA. The input nucleotidetriphosphate (NTP) mix is made in-house using natural and un-naturalNTPs.

A typical in vitro transcription reaction includes the following:

Template cDNA 1.0 μg 10x transcription buffer (400 mM Tris-HCl 2.0 μl pH8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) Custom NTPs (25 mM each7.2 μl RNase Inhibitor 20 U T7 RNA polymerase 3000 U dH₂O _(U)p to 20.0μl Incubation at 37° C for 3 hr-5 hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase is then used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA is purifiedusing Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA is quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

The T7 RNA polymerase may be selected from, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to, the novelpolymerases able to incorporate alternative NTPs as well as thosepolymerases described by Liu (Esvelt et al. (Nature (2011)472(7344):499-503 and U.S. Publication No. 20110177495) which recognizealternate promoters, Ellington (Chelliserrykattil and Ellington, NatureBiotechnology (2004) 22(9):1155-1160) describing a T7 RNA polymerasevariant to transcribe 2′-O-methyl RNA and Sousa (Padilla and Sousa,Nucleic Acids Research (2002) 30(24): e128) describing a T7 RNApolymerase double mutant; herein incorporated by reference in theirentireties.

B. Agarose Gel Electrophoresis of Alternative mRNA

Individual alternative mRNAs (200-400 ng in a 20 μl volume) are loadedinto a well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen,Carlsbad, Calif.) and run for 12-15 minutes according to themanufacturer protocol.

C. Agarose Gel Electrophoresis of RT-PCR Products

Individual reverse transcribed-PCR products (200-400 ng) are loaded intoa well of a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad,Calif.) and run for 12-15 minutes according to the manufacturerprotocol.

D. Nanodrop Alternative mRNA Quantification and UV Spectral Data

Alternative mRNAs in TE buffer (1 μl) are used for Nanodrop UVabsorbance readings to quantitate the yield of each alternative mRNAfrom an in vitro transcription reaction (UV absorbance traces are notshown).

Example 3. Enzymatic Capping of mRNA

Capping of the mRNA is performed as follows where the mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture is incubated at65° C. for 5 minutes to denature RNA, and then is transferredimmediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400 U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The mRNA is then purified using Ambion's MEGACLEAR™ Kit (Austin, Tex.)following the manufacturer's instructions. Following the cleanup, theRNA is quantified using the NANODROP™ (ThermoFisher, Waltham, Mass.) andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred. The RNA productmay also be sequenced by running a reverse-transcription-PCR to generatethe cDNA for sequencing.

Example 4. 5′-Guanosine Capping A. Materials and Methods

The cloning, gene synthesis and vector sequencing may be performed byDNA2.0 Inc. (Menlo Park, Calif.). The ORF is restriction digested usingXbaI and used for cDNA synthesis using tailed-or tail-less-PCR. Thetailed-PCR cDNA product is used as the template for the alternative mRNAsynthesis reaction using 25 mM each alternative nucleotide mix (allalternative nucleotides may be custom synthesized or purchased fromTriLink Biotech, San Diego, Calif. except pyrrolo-C triphosphate whichmay be purchased from Glen Research, Sterling Va.; unmodifed nucleotidesare purchased from Epicenter Biotechnologies, Madison, Wis.) andCellScript MEGASCRIPT™ (Epicenter Biotechnologies, Madison, Wis.)complete mRNA synthesis kit.

The in vitro transcription reaction is run for 4 hours at 37° C.Alternative mRNAs incorporating adenosine analogs are poly (A) tailedusing yeast Poly (A) Polymerase (Affymetrix, Santa Clara, Calif.). ThePCR reaction uses HiFi PCR 2× MASTER MIX™ (Kapa Biosystems, Woburn,Mass.). Alternative mRNAs are post-transcriptionally capped usingrecombinant Vaccinia Virus Capping Enzyme (New England BioLabs, Ipswich,Mass.) and a recombinant 2′-O-methyltransferase (EpicenterBiotechnologies, Madison, Wis.) to generate the 5′-guanosine Cap1structure. Cap 2 structure and Cap 2 structures may be generated usingadditional 2′-O-methyltransferases. The In vitro transcribed mRNAproduct is run on an agarose gel and visualized. Alternative mRNA may bepurified with Ambion/Applied Biosystems (Austin, Tex.) MEGAClear RNA™purification kit. The PCR uses PURELINK™ PCR purification kit(Invitrogen, Carlsbad, Calif.). The product is quantified on NANODROP™UV Absorbance (ThermoFisher, Waltham, Mass.). Quality, UV absorbancequality and visualization of the product was performed on an 1.2%agarose gel. The product is resuspended in TE buffer.

B. 5′-capping Alternative Nucleic Acid (mRNA) Structure

5′-capping of alternative mRNA may be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m⁷G(5′)ppp(5′)G (the ARCA cap);G(5′)ppp(5′)A; G(5′)ppp(5′)G; m⁷G(5′)ppp(5′)A; m⁷G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of alternative mRNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m⁷G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes are preferably derived from a recombinantsource.

When transfected into mammalian cells, the alternative mRNAs have astability of 12-18 hours or more than 18 hours, e.g., 24, 36, 48, 60, 72or greater than 72 hours.

Example 5. PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymeraseis preferably a recombinant enzyme expressed in yeast.

For studies performed and described herein, the poly-A tail is encodedin the IVT template to comprise 160 nucleotides in length. However, itshould be understood that the processivity or integrity of the poly-Atailing reaction may not always result in exactly 160 nucleotides. Hencepoly-A tails of approximately 160 nucleotides, acid about 150-165, 155,156, 157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scopeof the invention.

Example 6. In Vivo Expression of Selected Sequences

FIG. 1 shows in vivo expression data corresponding to control expressionof Target Protein 2 compared to the expression data for constructsgenerated using 4 novel codon sets (CO1, CO2, CO3 and CO4), afterintravenous administration of 0.05 mg/kg of each construct in MC3-LNP tomice. Similarly, FIG. 2 shows in vivo expression data corresponding tocontrol expression of Target Protein 2 compared to the expression datafor constructs generated using 6 novel codon sets (CO5, CO6, CO7, CO8,CO9 and CO10).

TABLE 11 Uracil Content of Selected Sequences Sequence Uracil ContentCO1 23% uracil CO2 27% uracil CO3 13% uracil + only 4 uracil pairs CO417% uracil CO7 13% uracil + only 4 uracil pairs CO9 14.7% uracil + only4 uracil pairs 

Example 7. Method of Screening for Protein Expression A. ElectrosprayIonization

A biological sample which may contain proteins encoded by alternativeRNA administered to the subject is prepared and analyzed according tothe manufacturer protocol for electrospray ionization (ESI) using 1, 2,3 or 4 mass analyzers. A biologic sample may also be analyzed using atandem ESI mass spectrometry system.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

B. Matrix-Assisted Laser Desorption/Ionization

A biological sample which may contain proteins encoded by alternativeRNA administered to the subject is prepared and analyzed according tothe manufacturer protocol for matrix-assisted laserdesorption/ionization (MALDI).

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

C. Liquid Chromatography-Mass Spectrometry-Mass Spectrometry

A biological sample, which may contain proteins encoded by alternativeRNA, may be treated with a trypsin enzyme to digest the proteinscontained within. The resulting peptides are analyzed by liquidchromatography-mass spectrometry-mass spectrometry (LC/MS/MS). Thepeptides are fragmented in the mass spectrometer to yield diagnosticpatterns that can be matched to protein sequence databases via computeralgorithms. The digested sample may be diluted to achieve 1 ng or lessstarting material for a given protein. Biological samples containing asimple buffer background (e.g., water or volatile salts) are amenable todirect in-solution digest; more complex backgrounds (e.g., detergent,non-volatile salts, glycerol) require an additional clean-up step tofacilitate the sample analysis.

Patterns of protein fragments, or whole proteins, are compared to knowncontrols for a given protein and identity is determined by comparison.

Example 8. Transfection A. Reverse Transfection

For experiments performed in a 24-well collagen-coated tissue cultureplate, Keratinocytes or other cells are seeded at a cell density of1×10⁵. For experiments performed in a 96-well collagen-coated tissueculture plate, Keratinocytes are seeded at a cell density of 0.5×10⁵.For each alternative mRNA to be transfected, alternative mRNA: RNAIMAX™are prepared as described and mixed with the cells in the multi-wellplate within 6 hours of cell seeding before cells had adhered to thetissue culture plate.

B. Forward Transfection

In a 24-well collagen-coated tissue culture plate, cells are seeded at acell density of 0.7×10⁵. For experiments performed in a 96-wellcollagen-coated tissue culture plate, keratinocytes, if used, are seededat a cell density of 0.3×10⁵. Cells are then grown to a confluencyof >70% for over 24 hours. For each alternative mRNA to be transfected,alternative mRNA: RNAIMAX™ are prepared as described and transfectedonto the cells in the multi-well plate over 24 hours after cell seedingand adherence to the tissue culture plate.

C. Translation Screen: ELISA

Cells are grown in EpiLife medium with Supplement S7 from Invitrogen ata confluence of >70%. Cells are reverse transfected with 300 ng of theindicated chemically alternative mRNA complexed with RNAIMAX™ fromInvitrogen. Alternatively, cells are forward transfected with 300 ngalternative mRNA complexed with RNAIMAX™ from Invitrogen. The RNA:RNAIMAX™ complex is formed by first incubating the RNA withSupplement-free EPILIFE® media in a 5× volumetric dilution for 10minutes at room temperature.

In a second vial, RNAIMAX™ reagent is incubated with Supplement-freeEPILIFE® Media in a 10× volumetric dilution for 10 minutes at roomtemperature. The RNA vial is then mixed with the RNAIMAX™ vial andincubated for 20-30 at room temperature before being added to the cellsin a drop-wise fashion. Secreted polypeptide concentration in theculture medium is measured at 18 hours post-transfection for each of thechemically alternative mRNAs in triplicate. Secretion of the polypeptideof interest from transfected human cells is quantified using an ELISAkit from Invitrogen or R&D Systems (Minneapolis, Minn.) following themanufacturer's recommended instructions.

D. Dose and Duration: ELISA

Cells are grown in EPILIFE® medium with Supplement S7 from Invitrogen ata confluence of >70%. Cells are reverse transfected with 0ng, 46.875ng,93.75ng, 187.5ng, 375ng, 750ng, or 1500ng alternative mRNA complexedwith RNAIMAX™ from Invitrogen. The alternative mRNA: RNAIMAX™ complex isformed as described. Secreted polypeptide concentration in the culturemedium is measured at 0, 6, 12, 24, and 48 hours post-transfection foreach concentration of each alternative mRNA in triplicate. Secretion ofthe polypeptide of interest from transfected human cells is quantifiedusing an ELISA kit from Invitrogen or R&D Systems following themanufacturers recommended instructions.

Example 9. Cellular Innate Immune Response: IFN-Beta ELISA and TNF-AlphaELISA

An enzyme-linked immunosorbent assay (ELISA) for Human Tumor NecrosisFactor-α (TNF-α), Human Interferon-β (IFN-β) and HumanGranulocyte-Colony Stimulating Factor (G-CSF) secreted from invitro-transfected Human Keratinocyte cells is tested for the detectionof a cellular innate immune response.

Cells are grown in EPILIFE® medium with Human Growth Supplement in theabsence of hydrocortisone from Invitrogen at a confluence of >70%. Cellsare reverse transfected with 0ng, 93.75ng, 187.5ng, 375ng, 750ng, 1500ngor 3000ng of the indicated chemically alternative mRNA complexed withRNAIMAX™ from Invitrogen as described in triplicate. Secreted TNF-α inthe culture medium is measured 24 hours post-transfection for each ofthe chemically alternative mRNAs using an ELISA kit from Invitrogenaccording to the manufacturer protocols.

Secreted IFN-β is measured 24 hours post-transfection for each of thealternative mRNAs using an ELISA kit from Invitrogen according to themanufacturer protocols. Secreted hu-G-CSF concentration is measured at24 hours post-transfection for each of the alternative mRNAs. Secretionof the polypeptide of interest from transfected human cells isquantified using an ELISA kit from Invitrogen or R&D Systems(Minneapolis, Minn.) following the manufacturers recommendedinstructions. These data indicate which alternative mRNA are capableeliciting a reduced cellular innate immune response in comparison tonatural and other alternative polynucleotides or reference compounds bymeasuring exemplary type 1 cytokines such as TNF-alpha and IFN-beta.

Example 10. Cytotoxicity and Apoptosis

This experiment demonstrates cellular viability, cytotoxity andapoptosis for distinct alternative mRNA-in vitro transfected HumanKeratinocyte cells. Keratinocytes are grown in EPILIFE® medium withHuman Keratinocyte Growth Supplement in the absence of hydrocortisonefrom Invitrogen at a confluence of >70%. Keratinocytes are reversetransfected with 0ng, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng, 1500ng,3000ng, or 6000ng of alternative mRNA complexed with RNAIMAX™ fromInvitrogen. The alternative mRNA: RNAIMAX™ complex is formed. SecretedhuG-CSF concentration in the culture medium is measured at 0, 6, 12, 24,and 48 hours post-transfection for each concentration of eachalternative mRNA in triplicate. Secretion of the polypeptide of interestfrom transfected human keratinocytes is quantified using an ELISA kitfrom Invitrogen or R&D Systems following the manufacturers recommendedinstructions. Cellular viability, cytotoxicity and apoptosis is measuredat 0, 12, 48, 96, and 192 hours post-transfection using the APOTOX-GLO™kit from Promega (Madison, Wis.) according to manufacturer instructions.

Example 11. In Vivo Assays with Human EPO Containing AlternativeNucleotides Formulation

Alternative hEPO mRNAs were formulated in lipid nanoparticles (LNPs)comprising DLin-KC2-DMA, DSPC, Cholesterol, and PEG-DMG at50:10:38.5:1.5 mol % respectively (Table 12). The LNPs were made bydirect injection utilizing nanoprecipitation of ethanol solubilizedlipids into a pH 4.0 50 mM citrate mRNA solution. The EPO LNP particlesize distributions were characterized by DLS. Encapsulation efficiency(EE) was determined using a Ribogreen™ fluorescence-based assay fordetection and quantification of nucleic acids.

TABLE 12 Formulation Conditions PEG Lipid Ionizable Lipid Phospholipid1,2-Dimyristoyl-sn- 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1,2-distearoyl-sn- glycerol, 1yl)-1,3-diocolan-4-yl)-N,N- glycero-3-Cholesterol methoxypolyethylene dimethylethanamine phosphocholinecholest-5-en-3β-ol Glycol (Lipid/Mol %) (Lipid/Mol %) (Lipid/Mol %)(Lipid/Mol %) DLin-KC2-DMA DSPC Cholesterol PEG-DMG 50 10 38.5 1.5

Methods and Data

Female Balb/c mice (n=5) were administered 0.05 mg/kg IM (50 μl in thequadriceps) or IV (100 μl in the tail vein) of human EPO mRNA. At time 8hours after the injection mice were euthanized and blood was collectedin serum separator tubes. The samples were spun, and serum samples werethen run on an EPO ELISA following the kit protocol (Stem CellTechnologies Catalog #01630).

Example 12. mRNA Sequences for Constructs Used to Screen Compounds ofthe Invention

hEPO DNA2.0 sequence (SEQ ID NO: 5):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGAGTGCACGAGTGTCCCGCGTGGTTGTGGTTGCTGCTGTCGCTCTTGAGCCTCCCACTGGGACTGCCTGTGCTGGGGGCACCACCCAGATTGATCTGCGACTCACGGGTACTTGAGAGGTACCTTCTTGAAGCCAAAGAAGCCGAAAACATCACAACCGGATGCGCCGAGCACTGCTCCCTCAATGAGAACATTACTGTACCGGATACAAAGGTCAATTTCTATGCATGGAAGAGAATGGAAGTAGGACAGCAGGCCGTCGAAGTGTGGCAGGGGCTCGCGCTTTTGTCGGAGGCGGTGTTGCGGGGTCAGGCCCTCCTCGTCAACTCATCACAGCCGTGGGAGCCCCTCCAACTTCATGTCGATAAAGCGGTGTCGGGGCTCCGCAGCTTGACGACGTTGCTTCGGGCTCTGGGCGCACAAAAGGAGGCTATTTCGCCGCCTGACGCGGCCTCCGCGGCACCCCTCCGAACGATCACCGCGGACACGTTTAGGAAGCTTTTTAGAGTGTACAGCAATTTCCTCCGCGGAAAGCTGAAATTGTATACTGGTGAAGCGTGTAGGACAGGGGATCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCTC TAGAhEPO CO9 sequence (SEQ ID NO: 6):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGCGTGCACGAGTGCCCCGCCTGGCTGTGGCTGCTGCTGAGCCTGCTGAGCCTGCCCCTGGGCCTGCCCGTGCTGGGCGCCCCCCCCCGCCTCATCTGCGACTCCCGCGTCCTCGAGCGCTACCTCCTCGAGGCCAAGGAGGCCGAGAACATCACCACCGGCTGCGCCGAGCACTGCTCCCTCAACGAGAACATCACCGTCCCCGACACCAAGGTCAACTTCTACGCCTGGAAGCGCATGGAGGTCGGCCAGCAGGCCGTCGAGGTCTGGCAGGGCCTCGCCCTCCTCTCCGAGGCCGTCCTCCGCGGCCAGGCCCTCCTCGTCAACTCCTCCCAGCCCTGGGAGCCCCTCCAGCTCCACGTCGACAAGGCCGTCTCCGGCCTCCGCTCCCTCACCACCCTCCTCCGCGCCCTCGGCGCCCAGAAGGAGGCCATCTCCCCCCCCGACGCCGCCTCCGCCGCCCCCCTCCGCACCATCACCGCCGACACCTTCCGCAAGCTCTTCCGCGTCTACTCCAACTTCCTCCGCGGCAAGCTCAAGCTCTACACCGGCGAGGCCTGCCGCACCGGCGACCGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGC GGCGCSF DNA2.0 sequence (SEQ ID NO: 7):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACACCTTTAGGACCTGCTTCTTCTTTACCTCAATCTTTTTTATTAAAATGTTTAGAACAAGTTAGAAAAATTCAAGGAGATGGAGCTGCTTTACAAGAAAAATTATGTGCTACATATAAATTATGTCATCCTGAAGAATTAGTTTTATTAGGACATTCTTTAGGAATTCCTTGGGCTCCTTTATCTTCTTGTCCTTCTCAAGCTTTACAATTAGCTGGATGTTTATCTCAATTACATTCTGGATTATTTTTATATCAAGGATTATTACAAGCTTTAGAAGGAATTTCTCCTGAATTAGGACCTACATTAGATACATTACAATTAGATGTTGCTGATTTTGCTACAACAATTTGGCAACAAATGGAAGAATTAGGAATGGCTCCTGCTTTACAACCTACACAAGGAGCTATGCCTGCTTTTGCTTCTGCTTTTCAAAGAAGAGCTGGAGGAGTTTTAGTTGCTTCTCATTTACAATCTTTTTTAGAAGTTTCTTATAGAGTTTTAAGACATTTAGCTCAACCTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC GCSF CO3 sequence (SEQ ID NO: 8):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACCCCCCTGGGCCCCGCCAGCAGCCTGCCCCAGAGCTTCCTGCTGAAGTGCCTGGAGCAGGTGCGGAAGATCCAGGGCGACGGCGCCGCCCTGCAGGAGAAGCTGTGCGCCACCTACAAGCTGTGCCACCCCGAGGAGCTGGTGCTGCTGGGCCACAGCCTGGGCATCCCCTGGGCCCCCCTGAGCAGCTGCCCCAGCCAGGCCCTGCAGCTGGCCGGCTGCCTGAGCCAGCTGCACAGCGGCCTGTTCCTGTACCAGGGCCTGCTGCAGGCCCTGGAGGGCATCAGCCCCGAGCTGGGCCCCACCCTGGACACCCTGCAGCTGGACGTGGCCGACTTCGCCACCACCATCTGGCAGCAGATGGAGGAGCTGGGCATGGCCCCCGCCCTGCAGCCCACCCAGGGCGCCATGCCCGCCTTCGCCAGCGCCTTCCAGCGGCGGGCCGGCGGCGTGCTGGTGGCCAGCCACCTGCAGAGCTTCCTGGAGGTGAGCTACCGGGTGCTGCGGCACCTGGCCCAGCCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCGCSF CO7 sequence (SEQ ID NO: 9):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACGCCGCTGGGGCCGGCGAGCAGCCTGCCGCAGAGCTTCCTGCTGAAGTGCCTGGAGCAGGTGAGGAAGATCCAGGGGGACGGGGCGGCGCTGCAGGAGAAGCTGTGCGCGACGTACAAGCTGTGCCACCCGGAGGAGCTGGTGCTGCTGGGGCACAGCCTGGGGATCCCGTGGGCGCCGCTGAGCAGCTGCCCGAGCCAGGCGCTGCAGCTGGCGGGGTGCCTGAGCCAGCTGCACAGCGGGCTGTTCCTGTACCAGGGGCTGCTGCAGGCGCTGGAGGGGATCAGCCCGGAGCTGGGGCCGACGCTGGACACGCTGCAGCTGGACGTGGCGGACTTCGCGACGACGATCTGGCAGCAGATGGAGGAGCTGGGGATGGCGCCGGCGCTGCAGCCGACGCAGGGGGCGATGCCGGCGTTCGCGAGCGCGTTCCAGAGGAGGGCGGGGGGGGTGCTGGTGGCGAGCCACCTGCAGAGCTTCCTGGAGGTGAGCTACAGGGTGCTGAGGCACCTGGCGCAGCCGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCGCSF CO9 sequence (SEQ ID NO: 10):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGCCGGCCCCGCCACCCAGAGCCCCATGAAGCTGATGGCCCTGCAGCTGCTGCTGTGGCACAGCGCCCTGTGGACCGTGCAGGAGGCCACCCCCCTCGGCCCCGCCTCCTCCCTCCCCCAGTCCTTCCTCCTCAAGTGCCTCGAGCAGGTCCGCAAGATCCAGGGCGACGGCGCCGCCCTCCAGGAGAAGCTCTGCGCCACCTACAAGCTCTGCCACCCCGAGGAGCTCGTCCTCCTCGGCCACTCCCTCGGCATCCCCTGGGCCCCCCTCTCCTCCTGCCCCTCCCAGGCCCTCCAGCTCGCCGGCTGCCTCTCCCAGCTCCACTCCGGCCTCTTCCTCTACCAGGGCCTCCTCCAGGCCCTCGAGGGCATCTCCCCCGAGCTCGGCCCCACCCTCGACACCCTCCAGCTCGACGTCGCCGACTTCGCCACCACCATCTGGCAGCAGATGGAGGAGCTCGGCATGGCCCCCGCCCTCCAGCCCACCCAGGGCGCCATGCCCGCCTTCGCCTCCGCCTTCCAGCGCCGCGCCGGCGGCGTCCTCGTCGCCTCCCACCTCCAGTCCTTCCTCGAGGTCTCCTACCGCGTCCTCCGCCACCTCGCCCAGCCCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Luc DNA2.0 sequence (SEQ ID NO: 11):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAGATGCGAAGAACATCAAGAAGGGACCTGCCCCGTTTTACCCTTTGGAGGACGGTACAGCAGGAGAACAGCTCCACAAGGCGATGAAACGCTACGCCCTGGTCCCCGGAACGATTGCGTTTACCGATGCACATATTGAGGTAGACATCACATACGCAGAATACTTCGAAATGTCGGTGAGGCTGGCGGAAGCGATGAAGAGATATGGTCTTAACACTAATCACCGCATCGTGGTGTGTTCGGAGAACTCATTGCAGTTTTTCATGCCGGTCCTTGGAGCACTTTTCATCGGGGTCGCAGTCGCGCCAGCGAACGACATCTACAATGAGCGGGAACTCTTGAATAGCATGGGAATCTCCCAGCCGACGGTCGTGTTTGTCTCCAAAAAGGGGCTGCAGAAAATCCTCAACGTGCAGAAGAAGCTCCCCATTATTCAAAAGATCATCATTATGGATAGCAAGACAGATTACCAAGGGTTCCAGTCGATGTATACCTTTGTGACATCGCATTTGCCGCCAGGGTTTAACGAGTATGACTTCGTCCCCGAGTCATTTGACAGAGATAAAACCATCGCGCTGATTATGAATTCCTCGGGTAGCACCGGTTTGCCAAAGGGGGTGGCGTTGCCCCACCGCACTGCTTGTGTGCGGTTCTCGCACGCTAGGGATCCTATCTTTGGTAATCAGATCATTCCCGACACAGCAATCCTGTCCGTGGTACCTTTTCATCACGGTTTTGGCATGTTCACGACTCTCGGCTATTTGATTTGCGGTTTCAGGGTCGTACTTATGTATCGGTTCGAGGAAGAACTGTTTTTGAGATCCTTGCAAGATTACAAGATCCAGTCGGCCCTCCTTGTGCCAACGCTTTTCTCATTCTTTGCGAAATCGACACTTATTGATAAGTATGACCTTTCCAATCTGCATGAGATTGCCTCAGGGGGAGCGCCGCTTAGCAAGGAAGTCGGGGAGGCAGTGGCCAAGCGCTTCCACCTTCCCGGAATTCGGCAGGGATACGGGCTCACGGAGACAACATCCGCGATCCTTATCACGCCCGAGGGTGACGATAAGCCGGGAGCCGTCGGAAAAGTGGTCCCCTTCTTTGAAGCCAAGGTCGTAGACCTCGACACGGGAAAAACCCTCGGAGTGAACCAGAGGGGCGAGCTCTGCGTGAGAGGGCCGATGATCATGTCAGGTTACGTGAATAACCCTGAAGCGACGAATGCGCTGATCGACAAGGATGGGTGGTTGCATTCGGGAGACATTGCCTATTGGGATGAGGATGAGCACTTCTTTATCGTAGATCGACTTAAGAGCTTGATCAAATACAAAGGCTATCAGGTAGCGCCTGCCGAGCTCGAGTCAATCCTGCTCCAGCACCCCAACATTTTCGACGCCGGAGTGGCCGGGTTGCCCGATGACGACGCGGGTGAGCTGCCAGCGGCCGTGGTAGTCCTCGAACATGGGAAAACAATGACCGAAAAGGAGATCGTGGACTACGTAGCATCACAAGTGACGACTGCGAAGAAACTGAGGGGAGGGGTAGTCTTTGTGGACGAGGTCCCGAAAGGCTTGACTGGGAAGCTTGACGCTCGCAAAATCCGGGAAATCCTGATTAAGGCAAAGAAAGGCGGGAAAATCGCTGTCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCLuc CO3 sequence (SEQ ID NO: 12):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGGACGCCAAGAACATCAAGAAGGGCCCCGCCCCCTTCTACCCCCTGGAGGACGGCACCGCCGGCGAGCAGCTGCACAAGGCCATGAAGCGGTACGCCCTGGTGCCCGGCACCATCGCCTTCACCGACGCCCACATCGAGGTGGACATCACCTACGCCGAGTACTTCGAGATGAGCGTGCGGCTGGCCGAGGCCATGAAGCGGTACGGCCTGAACACCAACCACCGGATCGTGGTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCCCGTGCTGGGCGCCCTGTTCATCGGCGTGGCCGTGGCCCCCGCCAACGACATCTACAACGAGCGGGAGCTGCTGAACAGCATGGGCATCAGCCAGCCCACCGTGGTGTTCGTGAGCAAGAAGGGCCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCCATCATCCAGAAGATCATCATCATGGACAGCAAGACCGACTACCAGGGCTTCCAGAGCATGTACACCTTCGTGACCAGCCACCTGCCCCCCGGCTTCAACGAGTACGACTTCGTGCCCGAGAGCTTCGACCGGGACAAGACCATCGCCCTGATCATGAACAGCAGCGGCAGCACCGGCCTGCCCAAGGGCGTGGCCCTGCCCCACCGGACCGCCTGCGTGCGGTTCAGCCACGCCCGGGACCCCATCTTCGGCAACCAGATCATCCCCGACACCGCCATCCTGAGCGTGGTGCCCTTCCACCACGGCTTCGGCATGTTCACCACCCTGGGCTACCTGATCTGCGGCTTCCGGGTGGTGCTGATGTACCGGTTCGAGGAGGAGCTGTTCCTGCGGAGCCTGCAGGACTACAAGATCCAGAGCGCCCTGCTGGTGCCCACCCTGTTCAGCTTCTTCGCCAAGAGCACCCTGATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCCAGCGGCGGCGCCCCCCTGAGCAAGGAGGTGGGCGAGGCCGTGGCCAAGCGGTTCCACCTGCCCGGCATCCGGCAGGGCTACGGCCTGACCGAGACCACCAGCGCCATCCTGATCACCCCCGAGGGCGACGACAAGCCCGGCGCCGTGGGCAAGGTGGTGCCCTTCTTCGAGGCCAAGGTGGTGGACCTGGACACCGGCAAGACCCTGGGCGTGAACCAGCGGGGCGAGCTGTGCGTGCGGGGCCCCATGATCATGAGCGGCTACGTGAACAACCCCGAGGCCACCAACGCCCTGATCGACAAGGACGGCTGGCTGCACAGCGGCGACATCGCCTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACCGGCTGAAGAGCCTGATCAAGTACAAGGGCTACCAGGTGGCCCCCGCCGAGCTGGAGAGCATCCTGCTGCAGCACCCCAACATCTTCGACGCCGGCGTGGCCGGCCTGCCCGACGACGACGCCGGCGAGCTGCCCGCCGCCGTGGTGGTGCTGGAGCACGGCAAGACCATGACCGAGAAGGAGATCGTGGACTACGTGGCCAGCCAGGTGACCACCGCCAAGAAGCTGCGGGGCGGCGTGGTGTTCGTGGACGAGGTGCCCAAGGGCCTGACCGGCAAGCTGGACGCCCGGAAGATCCGGGAGATCCTGATCAAGGCCAAGAAGGGCGGCAAGATCGCCGTGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC Luc CO7 sequence (SEQ ID NO: 13):TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGGACGCCAAGAACATCAAGAAGGGCCCCGCCCCCTTCTACCCCCTGGAGGACGGCACCGCCGGCGAGCAGCTGCACAAGGCCATGAAGAGGTACGCGCTGGTGCCGGGGACGATCGCGTTCACGGACGCGCACATCGAGGTGGACATCACGTACGCGGAGTACTTCGAGATGAGCGTGAGGCTGGCGGAGGCGATGAAGAGGTACGGGCTGAACACGAACCACAGGATCGTGGTGTGCAGCGAGAACAGCCTGCAGTTCTTCATGCCGGTGCTGGGGGCGCTGTTCATCGGGGTGGCGGTGGCGCCGGCGAACGACATCTACAACGAGAGGGAGCTGCTGAACAGCATGGGGATCAGCCAGCCGACGGTGGTGTTCGTGAGCAAGAAGGGGCTGCAGAAGATCCTGAACGTGCAGAAGAAGCTGCCGATCATCCAGAAGATCATCATCATGGACAGCAAGACGGACTACCAGGGGTTCCAGAGCATGTACACGTTCGTGACGAGCCACCTGCCGCCGGGGTTCAACGAGTACGACTTCGTGCCGGAGAGCTTCGACAGGGACAAGACGATCGCGCTGATCATGAACAGCAGCGGGAGCACGGGGCTGCCGAAGGGGGTGGCGCTGCCGCACAGGACGGCGTGCGTGAGGTTCAGCCACGCGAGGGACCCGATCTTCGGGAACCAGATCATCCCGGACACGGCGATCCTGAGCGTGGTGCCGTTCCACCACGGGTTCGGGATGTTCACGACGCTGGGGTACCTGATCTGCGGGTTCAGGGTGGTGCTGATGTACAGGTTCGAGGAGGAGCTGTTCCTGAGGAGCCTGCAGGACTACAAGATCCAGAGCGCGCTGCTGGTGCCGACGCTGTTCAGCTTCTTCGCGAAGAGCACGCTGATCGACAAGTACGACCTGAGCAACCTGCACGAGATCGCGAGCGGGGGGGCGCCGCTGAGCAAGGAGGTGGGGGAGGCGGTGGCGAAGAGGTTCCACCTGCCGGGGATCAGGCAGGGGTACGGGCTGACGGAGACGACGAGCGCGATCCTGATCACGCCGGAGGGGGACGACAAGCCGGGGGCGGTGGGGAAGGTGGTGCCGTTCTTCGAGGCGAAGGTGGTGGACCTGGACACGGGGAAGACGCTGGGGGTGAACCAGAGGGGGGAGCTGTGCGTGAGGGGGCCGATGATCATGAGCGGGTACGTGAACAACCCGGAGGCGACGAACGCGCTGATCGACAAGGACGGGTGGCTGCACAGCGGGGACATCGCGTACTGGGACGAGGACGAGCACTTCTTCATCGTGGACAGGCTGAAGAGCCTGATCAAGTACAAGGGGTACCAGGTGGCGCCGGCGGAGCTGGAGAGCATCCTGCTGCAGCACCCGAACATCTTCGACGCGGGGGTGGCGGGGCTGCCGGACGACGACGCGGGGGAGCTGCCGGCGGCGGTGGTGGTGCTGGAGCACGGGAAGACGATGACGGAGAAGGAGATCGTGGACTACGTGGCGAGCCAGGTGACGACGGCGAAGAAGCTGAGGGGGGGGGTGGTGTTCGTGGACGAGGTGCCGAAGGGGCTGACGGGGAAGCTGGACGCGAGGAAGATCAGGGAGATCCTGATCAAGGCGAAGAAGGGGGGGAAGATCGCGGTGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCmCherry wild-type (SEQ ID NO: 14)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTATCCAAGGGGGAGGAGGACAACATGGCGATCATCAAGGAGTTCATGCGATTCAAGGTGCACATGGAAGGTTCGGTCAACGGACACGAATTTGAAATCGAAGGAGAGGGTGAAGGAAGGCCCTATGAAGGGACACAGACCGCGAAACTCAAGGTCACGAAAGGGGGACCACTTCCTTTCGCCTGGGACATTCTTTCGCCCCAGTTTATGTACGGGTCCAAAGCATATGTGAAGCATCCCGCCGATATTCCTGACTATCTGAAACTCAGCTTTCCCGAGGGATTCAAGTGGGAGCGGGTCATGAACTTTGAGGACGGGGGTGTAGTCACCGTAACCCAAGACTCAAGCCTCCAAGACGGCGAGTTCATCTACAAGGTCAAACTGCGGGGGACTAACTTTCCGTCGGATGGGCCGGTGATGCAGAAGAAAACGATGGGATGGGAAGCGTCATCGGAGAGGATGTACCCAGAAGATGGTGCATTGAAGGGGGAGATCAAGCAGAGACTGAAGTTGAAAGATGGGGGACATTATGATGCCGAGGTGAAAACGACATACAAAGCGAAAAAGCCGGTGCAGCTTCCCGGAGCGTATAATGTGAATATCAAGTTGGATATTACTTCACACAATGAGGACTACACAATTGTCGAACAGTACGAACGCGCTGAGGGTAGACACTCGACGGGAGGCATGGACGAGTTGTACAAATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCmCherry CO3 sequence (SEQ ID NO: 15)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGGCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGAGCCCCCAGTTCATGTACGGCAGCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACCTGAAGCTGAGCTTCCCCGAGGGCTTCAAGTGGGAGCGGGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACAGCAGCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGGGGCACCAACTTCCCCAGCGACGGCCCCGTGATGCAGAAGAAGACCATGGGCTGGGAGGCCAGCAGCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGCGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCCGAGGTGAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTGAACATCAAGCTGGACATCACCAGCCACAACGAGGACTACACCATCGTGGAGCAGTACGAGCGGGCCGAGGGCCGGCACAGCACCGGCGGCATGGACGAGCTGTACAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGG CGGCmCherry CO7 sequence (SEQ ID NO: 16)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGGGAGGGGGAGGGGAGGCCGTACGAGGGGACGCAGACGGCGAAGCTGAAGGTGACGAAGGGGGGGCCGCTGCCGTTCGCGTGGGACATCCTGAGCCCGCAGTTCATGTACGGGAGCAAGGCGTACGTGAAGCACCCGGCGGACATCCCGGACTACCTGAAGCTGAGCTTCCCGGAGGGGTTCAAGTGGGAGAGGGTGATGAACTTCGAGGACGGGGGGGTGGTGACGGTGACGCAGGACAGCAGCCTGCAGGACGGGGAGTTCATCTACAAGGTGAAGCTGAGGGGGACGAACTTCCCGAGCGACGGGCCGGTGATGCAGAAGAAGACGATGGGGTGGGAGGCGAGCAGCGAGAGGATGTACCCGGAGGACGGGGCGCTGAAGGGGGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGGGGGCACTACGACGCGGAGGTGAAGACGACGTACAAGGCGAAGAAGCCGGTGCAGCTGCCGGGGGCGTACAACGTGAACATCAAGCTGGACATCACGAGCCACAACGAGGACTACACGATCGTGGAGCAGTACGAGAGGGCGGAGGGGAGGCACAGCACGGGGGGGATGGACGAGCTGTACAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC mCherry CO9 sequence (SEQ ID NO: 17)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGTGAGCAAGGGCGAGGAGGACAACATGGCCATCATCAAGGAGTTCATGCGGTTCAAGGTGCACATGGAGGGCAGCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTCAAGGTCACCAAGGGCGGCCCCCTCCCCTTCGCCTGGGACATCCTCTCCCCCCAGTTCATGTACGGCTCCAAGGCCTACGTCAAGCACCCCGCCGACATCCCCGACTACCTCAAGCTCTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTCATGAACTTCGAGGACGGCGGCGTCGTCACCGTCACCCAGGACTCCTCCCTCCAGGACGGCGAGTTCATCTACAAGGTCAAGCTCCGCGGCACCAACTTCCCCTCCGACGGCCCCGTCATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGCATGTACCCCGAGGACGGCGCCCTCAAGGGCGAGATCAAGCAGCGCCTCAAGCTCAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTCCAGCTCCCCGGCGCCTACAACGTCAACATCAAGCTCGACATCACCTCCCACAACGAGGACTACACCATCGTCGAGCAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTCTACAAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC

Example 13. Transfection in HeLa Cells

The day before transfection, 20,000 HeLa cells (ATCC no. CCL-2;Manassas, Va.) were harvested by treatment with Trypsin-EDTA solution(LifeTechnologies, Grand Island, N.Y.) and seeded in a total volume of100 μl EMEM medium (supplemented with 10% FCS and 1× Glutamax) per wellin a 96-well cell culture plate (Corning, Manassas, Va.). The cells weregrown at 37° C. in 5% CO₂ atmosphere overnight. Next day, 83ng ofLuciferase modRNA or 250ng of human GCSF modRNA, harboring chemicalalterations on the bases or the ribose units, were diluted in 10 μLfinal volume of OPTI-MEM (LifeTechnologies, Grand Island, N.Y.).Lipofectamine 2000 (LifeTechnologies, Grand Island, N.Y.) was used astransfection reagent and 0.2 μL were diluted in 10 μL final volume ofOPTI-MEM. After 5 min incubation at room temperature, both solutionswere combined and incubated additional 15 min at room temperature. Thenthe 20 μL were added to the 100 μL cell culture medium containing theHeLa cells. The plates were then incubated as described before. Fortransfection with mCherry or nanoLuc, a mixture of mRNA expressingmCherry or nanoLuc is mixed with 0.5 μL of Lipofectamine2000 (LifeTechnologies; cat#11668019) and OptiMem (Life Tehnologies;cat#31985062). A final volume of 20 μL of this mixture is added to 100μL of cells. The final amount of human EPO, G-CSF, Firefly Luciferase,mCherry and nanoLuc mRNA used per well is 250ng except for nanoLuc mRNAwhich we used at 25ng per well, respectively.

After 18 h to 22 h incubation, cells expressing luciferase were lysedwith 100 μl Passive Lysis Buffer (Promega, Madison, Wis.) according tomanufacturer instructions. Aliquots of the lysates were transferred towhite opaque polystyrene 96-well plates (Corning, Manassas, Va.) andcombined with 100 μL complete luciferase assay solution (Promega,Madison, Wis.). The lysate volumes were adjusted or diluted until nomore than 2 mio relative light units per well were detected for thestrongest signal producing samples. The background signal of the plateswithout reagent was about 200 relative light units per well. The platereader was a BioTek Synergy H1 (BioTek, Winooski, Vt.). The results areshown in Table 13.

For the cells transfected with mCherry, mCherry fluorescence reading wasmeasured directly of the cells at excitation of 585 nm and emission of615 nm wavelength. The results are shown in Table 14.

After 18 h to 22 h incubation, cell culture supernatants of cellsexpressing human EPO were collected and centrifuged at 10,000 rcf for 2min. The cleared supernatants were transferred and analyzed with a humanGCSF-specific or EPO ELISA kit (both from R&D Systems, Minneapolis,Minn.; Cat. #s SCS50, DEPOO, respectively) according to the manufacturerinstructions. All samples were diluted until the determined values werewithin the linear range of the human EPO ELISA standard curve. Theresults are shown in Table 15.

TABLE 13 Expression of FFLuc in HeLa cells FFLuc expression Construct(RLU; 24 hrs) 1-methyl pseudo U (DNA2.0) 78300 1-methyl pseudo U (CO7)115150 5-methoxy-uridine (CO3) 162900 5-methoxy-uridine (CO7) 685505-methoxy-uridine (CO9) 93150

TABLE 14 Expression of mCherry in HeLa cells mCherry expressionConstruct (FLU; 24 hrs) 1-methyl pseudo U (WT) 1137 1-methyl pseudo U(CO7) 2229 5-methoxy-uridine (CO3) 1464 5-methoxy-uridine (CO7) 30075-methoxy-uridine (CO9) 4344

TABLE 15 Expression of hEPO in HeLa cells hEPO expression in HeLaConstruct (mIU/mL; 24 hours) 1-methyl pseudo U (DNA2.0) 2505375-methoxy-uridine (CO3) 253718 5-methoxy-uridine (CO7) 2909255-methoxy-uridine (CO9) 123977

Example 14. Transfection in BJ Fibroblasts

At 2 or 3 days prior to transfection, 100,000 BJ fibroblast cells (ATCCno. CRL-2522; Manassas, Va.) were harvested by treatment withtrypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seededin a total volume of 500 μL EMEM medium (supplemented with 10% FCS and10% Glutamax, both LifeTechnologies, Grand Island, N.Y.) per well in24-well cell culture plates (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO₂ atmosphere overnight. On the next day, 500ng modRNA, harboring chemical alterations on the bases or the riboseunits, were diluted in 25 μL final volume of OPTI-MEM (LifeTechnologies,Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island,N.Y.) was used as transfection reagent and 1.0 μL was diluted in 25 μLfinal volume of OPTI-MEM. After 5 min incubation at room temperature,both solutions were combined and incubated an additional 15 min at roomtemperature. The 50 μL were added to the 500 μL cell culture mediumcontaining the BJ fibroblast cells. The plates were then incubated asdescribed above.

After 18 h to 22 h incubation, cell culture supernatants of cellsexpressing human GCSF or human EPO were collected and centrifuged at10,000 rcf for 2 min. The cleared supernatants were transferred andanalyzed with a human GCSF-specific or EPO ELISA kit (both from R&DSystems, Minneapolis, Minn.; Cat. #s SCS50, DEPOO, respectively)according to the manufacturer instructions. All samples were diluteduntil the determined values were within the linear range of the humanGCSF or EPO ELISA standard curve. The results are shown in Tables 16,17, and 18.

TABLE 16 Expression of hEPO in BJ Fibroblast cells hEPO expression in BJConstruct (mIU/mL; 48 hours) 1-methyl pseudo U (DNA2.0) 1537135-methoxy-uridine (CO3) 146050 5-methoxy-uridine (CO7) 1581955-methoxy-uridine (CO9) 68986

TABLE 17 Expression of GCSF in BJ Fibroblast cells (25 ng/well) GCSFexpression in BJ Construct (pg/mL; 48 hours) 1-methyl pseudo U (DNA2.0)153172 5-methoxy-uridine (CO3) 366060 5-methoxy-uridine (CO7) 1907765-methoxy-uridine (CO9) 119084

TABLE 18 Expression of GCSF in BJ Fibroblast cells (15 ng/well) GCSFexpression in BJ Construct (pg/mL; 48 hours) 1-methyl pseudo U (DNA2.0)357902 5-methoxy-uridine (CO3) 766241 5-methoxy-uridine (CO7) 5558145-methoxy-uridine (CO9) 330441

Example 15. Cytokine Screen in BJ Fibroblast Cells

At 2 or 3 days prior to transfection, 100,000 BJ fibroblast cells (ATCCno. CRL-2522; Manassas, Va.) were harvested by treatment withtrypsin-EDTA solution (LifeTechnologies, Grand Island, N.Y.) and seededin a total volume of 500 ul EMEM medium (supplemented with 10% FCS and10% Glutamax, both LifeTechnologies, Grand Island, N.Y.) per well in24-well cell culture plates (Corning, Manassas, Va.). The cells weregrown at 37° C. in a 5% CO₂ atmosphere overnight. On the next day, 500ng modRNA, harboring chemical alterations on the bases or the riboseunits, were diluted in 25 μL final volume of OPTI-MEM (LifeTechnologies,Grand Island, N.Y.). Lipofectamine 2000 (LifeTechnologies, Grand Island,N.Y.) was used as transfection reagent and 1.0 μL was diluted in 25 μLfinal volume of OPTI-MEM. After 5 min incubation at room temperature,both solutions were combined and incubated an additional 15 min at roomtemperature. The 50 μL were added to the 500 μL cell culture mediumcontaining the BJ fibroblast cells. The plates were then incubated asdescribed above.

After 18 h to 22 h incubation, cell culture supernatants were collectedand centrifuged at 10,000 rcf for 2 min. The cleared supernatants weretransferred and analyzed with a human IFN-beta ELISA (R&D Systems,Minneapolis, Minn.; Cat. #s 41410-2) and human CCL-5/RANTES ELISA (R&DSystems, Minneapolis, Minn.; Cat. #s SRNOOB) according to themanufacturer instructions. All samples were diluted until the determinedvalues were within the linear range of the ELISA standard curves using aBioTek Synergy H1 μlate reader (BioTek, Winooski, Vt.). The results areshown in Tables 19, 20, and 21.

TABLE 19 INFβ expression in BJ Fibroblast cells by GCSF mRNA IFN-bexpression in BJ Construct (pg/mL; 48 hours) 1-methyl pseudo U (DNA2.0)20 5-methoxy-uridine (CO3) 0 5-methoxy-uridine (CO7) 0 5-methoxy-uridine(CO9) 0

TABLE 20 INFβ expression in BJ Fibroblast cells by FFLuc mRNA IFN-bexpression in BJ Construct (pg/mL; 48 hours) 1-methyl pseudo U (CO7) 1945-methoxy-uridine (CO3) 0 5-methoxy-uridine (CO7) 0 5-methoxy-uridine(CO9) 0

TABLE 21 INFβ expression in BJ Fibroblast cells by mCherry mRNA IFN-bexpression in BJ Construct (pg/mL; 48 hours) 1-methyl pseudo U (CO7) 3095-methoxy-uridine (CO3) 0 5-methoxy-uridine (CO7) 0 5-methoxy-uridine(CO9) 0

Example 16. In Vivo Expression of mRNA

Using the method described in Example 11, in vivo expression of thealternative mRNA of Example 12 was studied. Female CD-1 mice wereadministered the mRNAs intravenously at 0.05 mg/kg. The results areshown in Tables 22, 23, and 24.

TABLE 22 In vivo expression of GCSF Dose group (0.05 mg/kg) GCSF @ 6hours (ng/mL) 1-methyl pseudo (WT) 71.4 5-methoxy-uridine (CO3) 32.55-methoxy-uridine (CO7) 8.6 5-methoxy-uridine (CO9) 30.7

TABLE 23 In vivo expression of Luciferase Dose group (0.05 mg/kg) Totalflux @ 6 hours (RLU) 1-methyl pseudo (DNA2.0) 2.38 × 10⁸ 1-methyl pseudo(CO7) 1.40 × 10⁹ 5-methoxy-uridine (CO3) 5.26 × 10⁸ 5-methoxy-uridine(CO7) 1.86 × 10⁸

TABLE 24 In vivo expression of GCSF GCSF @ GCSF @ GCSF @ 3 hours 6 hours24 hours Dose group (0.05 mg/kg) (ng/mL) (ng/mL) (ng/mL) 1-methylpseudoU (DNA2.0) 409.6 546.0 168.2 1-methyl pseudoU (CO3) 517.9 637.4274.0 1-methyl pseudoU (CO7) 355.8 473.5 220.2 1-methyl pseudoU (CO9)547.7 726.3 124.5 5-methoxy-uridine (CO3) 234.1 277.8 64.65-methoxy-uridine (CO7) 308.6 341.9 83.3 5-methoxy-uridine (CO9) 253.9285.3 51.9

Example 17. In Vivo Expression of mRNA in Non-Human Primates

Cynomolgus monkeys are administered a standard MC3 formulation includingan mRNA encoding hEPO. The expression of hEPO was measured using anELISA method before and 2, 6, 24, 48, 72, 96, and 120 hours afteradministration. Male monkeys were administered the formulation at a doserate of 5 mL/kg/h for 1 hour.

TABLE 25 In vivo expression of hEPO Dose group (0.05 mg/kg) hEPO Cmax(ng/mL) AUC (hr*pg/mL) 1-methyl pseudo (DNA2.0) 70.0 9549545-methoxy-uridine (CO9) 50.6 984832

TABLE 26 In vivo expression of hEPO hEPO @ hEPO @ hEPO @ 6 hours 12hours 24 hours Dose group (0.05 mg/kg) (ng/mL) (ng/mL) (ng/mL) 1-methylpseudoU (DNA2.0) 72.7 14.7 2.1 5-methoxy-uridine (CO9) 87.1 62.6 18.9

Example 18. mRNA-Templated In Vitro Transcription

Human Epo 1-methylpseudouridine-containing mRNA was produced by run-offin vitro transcription using standard 4 h plasmid-based IVT reactionconditions. The material was subjected to reverse phase purification,and the INF-β clear fractions were pooled. From this pooled material, astandard 1-methylpseudouridine-containing and5-methoxy-uridine-containing 4 h plasmid-based IVT reaction was run butin place of DNA template, INF-β clear mRNA was added to a finalconcentration of 1 mg/mL. After 4 h, the reaction was split in two, withpart being used for LC analysis and part to be transfected into BJfibroblasts using L-2000 according to the manufacturer suggestedprotocol. After 48 hours, the presence of INF-β was determined by ELISA.LC analysis showed the presence of n+1 polymers to be in much higherabundance in the samples incubated with 1-methylpseudouridine-containingnucleotides compared to 5-methoxy-uridine-containing nucleotides.Additionally, the 1-methylpseudouridine-containing sample showedsignificantly more INF-β response compared to the5-methoxy-uridine-containing sample.

Example 19. In Vitro Transcription Temperature Dependence

Human Epo 1-methylpseudouridine-containing and5-methoxy-uridine-containing mRNA was produced by run-off in vitrotranscription using our standard 4 h plasmid-based IVT reactionconditions. The mRNA was split and part was subjected to oligo dTpurification whereas the other part was crude reaction mixture. Bothwere transfected into BJ fibroblasts using L-2000, and INF-β levels weredetermined by ELISA. The dT purified and crude5-methoxy-uridine-containing mRNA showed marginal INF-β whether the IVTwas performed at 25° C. or 37° C., whereas the1-methylpseudouridine-containing mRNA showed significant increases inINF-β induction at 25° C. compared to 37° C. The results are shown inFIG. 3.

Example 20. Production of mRNA with a 20 Consecutive Uridine Tail

A reverse PCR primer was designed to code for an mRNA with a tailstructure of 100A20U-3′. PCR was completed as previously described, andrun-off IVT was performed according to PCR-templated 4 h IVT conditionsunder either 1-methylpseudouridine-containing mRNA or5-methoxy-uridine-containing mRNA conditions. The IVT was reverse-phasepurified, the fractions were diafiltered into water, and transfectedinto BJ fibroblasts using L-2000. After 48 hours, INF-β levels weredetermined by ELISA. The results are shown in FIG. 4.

Other Embodiments

It is to be understood that while the present disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the present disclosure, which is defined by the scope of the appendedclaims. Other aspects, advantages, and alterations are within the scopeof the following claims.

1-27. (canceled)
 28. A pharmaceutical composition comprising an mRNAencoding a polypeptide, and a pharmaceutically acceptable excipient,wherein the mRNA comprises: (i) a 5′-cap structure; (ii) a 5′-UTR; (iii)an open reading frame (ORF) encoding the polypeptide, wherein at least95% of uracils in the ORF are 5-methoxyuracils; and wherein the uracilcontent in the ORF is between the theoretical minimum and 150% of thetheoretical minimum, (iv) a 3′-UTR; and (v) a poly-A region; wherein thelevel of expression in a mammalian cell of the encoded polypeptide fromthe mRNA is increased relative to a reference mRNA comprising areference open reading frame (rORF) encoding the polypeptide, wherein atleast 95% of uracils in the rORF are 5-methoxyuracils, and wherein theuracil content in the rORF is from about 190% to about 200% of thetheoretical minimum.
 29. The pharmaceutical composition of claim 28,wherein the uracil content in the ORF is between the theoretical minimumand 125% of the theoretical minimum.
 30. The pharmaceutical compositionof claim 28, wherein the guanine content of the ORF is maximized for atleast 50% of the codons, and wherein the ORF comprises at least one lowfrequency guanine maximized codon.
 31. The pharmaceutical composition ofclaim 28, wherein the cytosine content of the ORF is maximized for atleast 50% of the codons, and wherein the ORF comprises at least one lowfrequency cytosine maximized codon.
 32. The pharmaceutical compositionof claim 28, wherein the guanine and cytosine content of the ORF ismaximized for at least 50% of the codons, and wherein the ORF comprisesat least one low frequency guanine and cytosine maximized codon.
 33. Thepharmaceutical composition of claim 28, wherein the uracil content isless than 20% of the total nucleobase content in the ORF.
 34. Thepharmaceutical composition of claim 28, wherein the uracil contentwithin any 20 nucleobase window within the ORF does not exceed 50%. 35.The pharmaceutical composition of claim 28, wherein the 5′-cap structureis cap0, cap1, or ARCA inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, or 2-azido-guanosine.
 36. Thepharmaceutical composition of claim 28, wherein the 3′-UTR is analpha-globin 3′-UTR.
 37. The pharmaceutical composition of claim 28,wherein the poly-A region is between 80 to 120 nucleotides in length.38. The pharmaceutical composition of claim 28, wherein, upon contactinga mammalian cell, the mRNA has a longer half-life, greater area underthe curve of protein expression, or both, relative to a correspondingwild-type mRNA.
 39. The pharmaceutical composition of claim 28, whereinthe ORF further comprises at least one low-frequency codon.
 40. Thepharmaceutical composition of claim 28, wherein the 3′-UTR of the mRNAcomprises at least one microRNA (miRNA) binding site.
 41. Thepharmaceutical composition of claim 40, wherein the miRNA is known to beexpressed in liver cells or in immune cells.
 42. The pharmaceuticalcomposition of claim 41, wherein the miRNA is chosen from miR-142-5p,miR-146-5p, or miR-146-3p.
 43. The pharmaceutical composition of claim41, wherein the miRNA is miR-142-3p.
 44. The pharmaceutical compositionof claim 40, wherein the miRNA is known to be expressed in liver cells,and is miR-122-5p or miR-122-3p.
 45. The pharmaceutical composition ofclaim 1, wherein the composition is in a single-unit dosage form. 46.The pharmaceutical composition of claim 1, wherein the composition is ina multi-unit dosage form.
 47. The pharmaceutical composition of claim 1,wherein the mRNA is purified.
 48. The pharmaceutical composition ofclaim 47, wherein the mRNA is purified without reverse phasechromatography.